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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 28 Apr 2025 at 01:42 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: ( "CRISPR.CAS" OR "crispr/cas" ) NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

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RevDate: 2025-04-27
CmpDate: 2025-04-27

Chen X, Yao S, Xie L, et al (2025)

Disruption of the odorant receptor co-receptor (Orco) reveals its critical role in multiple olfactory behaviors of a cosmopolitan pest.

Insect biochemistry and molecular biology, 177:104248.

The olfactory system of insects plays a pivotal role in multiple, essential activities including feeding, mating, egg laying, and host localization. The capacity of odorant receptors to recognize odor molecules relies on odorant receptor co-receptors forming heterodimers. Here we report the successful engineering a homozygous mutant strain of diamondback moth (Plutella xylostella) in which the odorant receptor co-receptor PxOrco was silenced using CRISPR/Cas9. This insect is a globally important crop pest for which novel control methods are urgently required. Behavioral assays demonstrated that PxOrco knockout males exhibited abolished courtship behaviors, inability to mate, and loss of selective preference for P. xylostella's key sex pheromone components. Whilst female mating behavior and fecundity remained unaffected by PxOrco knockout, oviposition response to leaf alcohol, a key cue for normal oviposition behavior, was lost. Electroantennography revealed drastically reduced responses to sex pheromones and plant volatiles in PxOrco-deficient adults but food location by larvae was unaffected. Moreover, expression analysis of PxOrco-deficient pheromone receptors (PRs) indicated varied regulation patterns, with down-regulation observed in several PRs in both sexes. These findings underscore the critical role of PxOrco in regulating multiple olfactory aspects in P. xylostella, including feeding, mating, and host location. Our study identifies the potential of disrupting the Orco gene in this and other pest species to provide novel avenues for future pest control.

RevDate: 2025-04-27
CmpDate: 2025-04-27

Lou H, Li S, Shi Z, et al (2025)

Engineering source-sink relations by prime editing confers heat-stress resilience in tomato and rice.

Cell, 188(2):530-549.e20.

A 2°C climate-warming scenario is expected to further exacerbate average crop losses by 3%-13%, yet few heat-tolerant staple-crop varieties are available toward meeting future food demands. Here, we develop high-efficiency prime-editing tools to precisely knockin a 10-bp heat-shock element (HSE) into promoters of cell-wall-invertase genes (CWINs) in elite rice and tomato cultivars. HSE insertion endows CWINs with heat-responsive upregulation in both controlled and field environments to enhance carbon partitioning to grain and fruits, resulting in per-plot yield increases of 25% in rice cultivar Zhonghua11 and 33% in tomato cultivar Ailsa Craig over heat-stressed controls, without fruit quality penalties. Up to 41% of heat-induced grain losses were rescued in rice. Beyond a prime-editing system for tweaking gene expression by efficiently delivering bespoke changes into crop genomes, we demonstrate broad and robust utility for targeted knockin of cis-regulatory elements to optimize source-sink relations and boost crop climate resilience.

RevDate: 2025-04-27
CmpDate: 2025-04-27

Zhang Y, David NL, Pesaresi T, et al (2024)

Noncoding variation near UBE2E2 orchestrates cardiometabolic pathophenotypes through polygenic effectors.

JCI insight, 10(2):.

Mechanisms underpinning signals from genome-wide association studies remain poorly understood, particularly for noncoding variation and for complex diseases such as type 2 diabetes mellitus (T2D) where pathogenic mechanisms in multiple different tissues may be disease driving. One approach is to study relevant endophenotypes, a strategy we applied to the UBE2E2 locus where noncoding single nucleotide variants (SNVs) are associated with both T2D and visceral adiposity (a pathologic endophenotype). We integrated CRISPR targeting of SNV-containing regions and unbiased CRISPR interference (CRISPRi) screening to establish candidate cis-regulatory regions, complemented by genetic loss of function in murine diet-induced obesity or ex vivo adipogenesis assays. Nomination of a single causal gene was complicated, however, because targeting of multiple genes near UBE2E2 attenuated adipogenesis in vitro; CRISPR excision of SNV-containing noncoding regions and a CRISPRi regulatory screen across the locus suggested concomitant regulation of UBE2E2, the more distant UBE2E1, and other neighborhood genes; and compound heterozygous loss of function of both Ube2e2 and Ube2e1 better replicated pathological adiposity and metabolic phenotypes compared with homozygous loss of either gene in isolation. This study advances a model whereby regulatory effects of noncoding variation not only extend beyond the nearest gene but may also drive complex diseases through polygenic regulatory effects.

RevDate: 2025-04-27
CmpDate: 2025-04-27

Moffa L, Mannino G, Bevilacqua I, et al (2025)

CRISPR/Cas9-driven double modification of grapevine MLO6-7 imparts powdery mildew resistance, while editing of NPR3 augments powdery and downy mildew tolerance.

The Plant journal : for cell and molecular biology, 122(2):e17204.

The implementation of genome editing strategies in grapevine is the easiest way to improve sustainability and resilience while preserving the original genotype. Among others, the Mildew Locus-O (MLO) genes have already been reported as good candidates to develop powdery mildew-immune plants. A never-explored grapevine target is NPR3, a negative regulator of the systemic acquired resistance. We report the exploitation of a cisgenic approach with the Cre-lox recombinase technology to generate grapevine-edited plants with the potential to be transgene-free while preserving their original genetic background. The characterization of three edited lines for each target demonstrated immunity development against Erysiphe necator in MLO6-7-edited plants. Concomitantly, a significant improvement of resilience, associated with increased leaf thickness and specific biochemical responses, was observed in defective NPR3 lines against E. necator and Plasmopara viticola. Transcriptomic analysis revealed that both MLO6-7 and NPR3 defective lines modulated their gene expression profiles, pointing to distinct though partially overlapping responses. Furthermore, targeted metabolite analysis highlighted an overaccumulation of stilbenes coupled with an improved oxidative scavenging potential in both editing targets, likely protecting the MLO6-7 mutants from detrimental pleiotropic effects. Finally, the Cre-loxP approach allowed the recovery of one MLO6-7 edited plant with the complete removal of transgene. Taken together, our achievements provide a comprehensive understanding of the molecular and biochemical adjustments occurring in double MLO-defective grape plants. In parallel, the potential of NPR3 mutants for multiple purposes has been demonstrated, raising new questions on its wide role in orchestrating biotic stress responses.

RevDate: 2025-04-25
CmpDate: 2025-04-25

Chen Y, Li M, Liu X, et al (2025)

Establishment of CRISPR-STAR System to Realise Simultaneous Transcriptional Activation and Repression in Yarrowia lipolytica.

Microbial biotechnology, 18(4):e70151.

The ability to regulate gene expression in multiple directions is crucial to maximise the production of microbial cell factories. However, the lack of a regulatory tool that can simultaneously activate and repress multiple genes restricts the manipulation diversity of Yarrowia lipolytica, which is an industrial workhorse for bioproduction. To address this issue, we developed a CRISPR scaffold RNAs (scRNAs)-mediated transcriptional activation and repression (CRISPR-STAR) platform. Firstly, we evaluated different methods for bidirectional regulation using CRISPR on both endogenous and synthetic promoters in Y. lipolytica, and chose the utilisation of orthogonal scRNAs to recruit activation and inhibition domains. Secondly, CRISPR-STAR was optimised by the introduction of alternative dCas proteins, scRNA structures and activators. 2.6-fold and 54.9-fold activation were achieved for synthetic and endogenous promoters, respectively, when the VPR transcriptional activator was recruited via MS2 hairpin. The repression of several genes was successfully achieved, with repression levels ranging from 3% to 32%, when the MXI1 transcriptional repressor was recruited via PP7 hairpin. Finally, CRISPR-STAR was applied to enhance fatty alcohol production by activating the FAR gene (encodes fatty acyl-CoA reductase) and repression of the PEX10 gene (encodes an integral membrane protein required for peroxisome biogenesis and matrix protein import). Compared to the non-targeting control, the bidirectionally regulated strain showed a 55.7% increase in yield to 778.8 mg/L. Our findings demonstrate that the CRISPR-STAR platform enables multi-mode regulation of genes, offering engineering opportunities to improve the productive performance of Y. lipolytica.

RevDate: 2025-04-25
CmpDate: 2025-04-25

Kelly G, Plesser E, Bdolach E, et al (2025)

In planta genome editing in citrus facilitated by co-expression of CRISPR/Cas and developmental regulators.

The Plant journal : for cell and molecular biology, 122(2):e70155.

Recent advances in the field of genome editing offer a promising avenue for targeted trait improvements in fruit trees. However, the predominant method taken for genome editing in citrus (and other fruit trees) involves the time-consuming tissue culture approach, thereby prolonging the overall citrus breeding process and subjecting it to the drawbacks associated with somaclonal variation. In this study, we introduce an in planta approach for genome editing in soil-grown citrus plants via direct transformation of young seedlings. Our editing system, abbreviated here as IPGEC (in planta genome editing in citrus), is designed to transiently co-express three key gene groups in citrus tissue via Agrobacterium tumefaciens: (i) a genome-editing catalytic group, (ii) a shoot induction and regeneration group, and (iii) a T-DNA enhanced delivery group. This integrated system significantly improves de novo shoot induction and regeneration efficiency of edited tissue. By incorporating single-guides RNA's (sgRNA's) targeting the carotenoid biosynthetic gene PHYTOENE DESATURASE (CsPDS), the IPGEC system effectively produced mutated albino shoots, confirming its ability to generate homozygous/biallelic genome-edited plants. By using high throughput screening, we provide evidence that transgene-free genome-edited plants could be obtained following the IPGEC approach. Our findings further suggest that the efficiency of specific developmental regulators in inducing transformation and regeneration rates may be cultivar-specific and therefore needs to be optimized per cultivar. Finally, targeted breeding for specific trait improvements in already successful cultivars is likely to revolutionize fruit tree breeding and will pave the way for accelerating the development of high-quality citrus cultivars.

RevDate: 2025-04-24
CmpDate: 2025-04-25

Yang Z, Bai W, Guo G, et al (2025)

The Q-interacted protein QIP3 recruits TaTPL to regulate spike architecture in wheat.

The Plant journal : for cell and molecular biology, 122(2):e70149.

Spike architecture is a critical determinant of grain yield in wheat; yet the regulatory mechanisms remain poorly understood. Here, we demonstrate that the AP2 transcription factor Q directly represses the expression of TaMYB30-6A, a gene associated with spike length in wheat. We further identify QIP3 as a Q-interacting protein harboring an N-terminal EAR motif. Simultaneously, we reveal that QIP3 exhibits transcriptional repression activity, dependent on the EAR motif, and physically interacts with the transcriptional corepressor TaTPL. Importantly, the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-generated qip3-aabbdd mutants exhibit reduced plant height and increased spike length phenotypes. Furthermore, RNA-seq and RT-qPCR assays show that QIP3 negatively regulates the expression of the Q target gene TaMYB30-6A in wheat. Collectively, we propose that the EAR motif-containing QIP3 interacts with Q to regulate spike architecture by recruiting the transcriptional corepressor TaTPL in wheat.

RevDate: 2025-04-26
CmpDate: 2025-04-25

Zhang Z, Abreu B, Brothwood JL, et al (2025)

The identification of functional regions of MEK1 using CRISPR tiling screens.

Communications biology, 8(1):656.

CRISPR tiling screen is a powerful tool to identify protein regions relevant to its biological function. Understanding the functional relevance of the regions of target protein is of great help for structure-based drug discovery. Studying the drug resistance mechanisms of small-molecule inhibitors is important for the development and clinical application of the compounds. Using MEK1 and MEK inhibitors as example here, we demonstrate the utility of CRISPR tiling to identify regions essential for cancer cell viability and regions where mutations are resistant to MEK inhibitors. We study the drug resistance mechanisms of the regions and discussed the potential, as well as limitations, of applying the technology to drug development. Our findings demonstrate the value and prompt the utilization of CRISPR tiling technology in structure-based drug discovery.

RevDate: 2025-04-26
CmpDate: 2025-04-25

Chai R, Guo J, Yang C, et al (2025)

Enhanced chemotaxis and degradation of nonylphenol in Pseudoxanthomonas mexicana via CRISPR-mediated receptor modification.

Scientific reports, 15(1):14296.

In this study, a novel nonylphenol (NP)-degrading bacterium, Pseudoxanthomonas mexicana CH, was isolated from wastewater treatment plant effluent. Phylogenetic analysis showed its close relationship to P. mexicana AMX 26BT. The strain displayed chemotaxis toward NP, with Mcp24 as the key chemoreceptor. The Mcp24 deletion mutant (CH- 1) had weaker chemotaxis and NP degradation (over 30% lower in solution and 8% lower in sludge than the wild type). In vitro, Mcp15's C-terminal pentapeptide DWQEF was methylated by CheR. Using CRISPR, this pentapeptide was added to Mcp24 to create CH- 2. CH- 2 showed better NP chemotaxis (17% higher in plate assays and 39% higher in capillary assays) and higher NP degradation rates (23.5% and 24.2% higher in solution and sludge, respectively). These findings demonstrate that NP acts as a bacterial chemoattractant, with Mcp24 as the receptor. Enhancing Mcp24's C-terminal pentapeptide improves chemotaxis and degradation efficiency, representing a significant advancement in bioremediation by strengthening bacterial responses to pollutants.

RevDate: 2025-04-26
CmpDate: 2025-04-25

Balakrishnan A, Hunziker M, Tiwary P, et al (2025)

A CRISPR homing screen finds a chloroquine resistance transporter-like protein of the Plasmodium oocyst essential for mosquito transmission of malaria.

Nature communications, 16(1):3895.

Genetic screens with barcoded PlasmoGEM vectors have identified thousands of Plasmodium berghei gene functions in haploid blood stages, gametocytes and liver stages. However, the formation of diploid cells by fertilisation has hindered similar research on the parasites' mosquito stages. In this study, we develop a scalable genetic system that uses barcoded gene targeting vectors equipped with a CRISPR-mediated homing mechanism to generate homozygous loss-of-function mutants after one parent introduces a modified allele into the zygote. To achieve this, we use vectors additionally expressing a target gene specific gRNA. When integrated into one of the parental alleles it directs Cas9 to the intact allele after fertilisation, leading to its disruption. This homing strategy is 90% effective at generating homozygous gene editing of a fluorescence-tagged reporter locus in the oocyst. A pilot screen identifies PBANKA_0916000 as a chloroquine resistance transporter-like protein (CRTL) essential for oocyst growth and sporogony, pointing to an unexpected importance for malaria transmission of the poorly understood digestive vacuole of the oocyst that contains hemozoin granules. Homing screens provide a method for the systematic discovery of malaria transmission genes whose first essential functions are after fertilisation in the bloodmeal, enabling their potential as targets for transmission-blocking interventions to be assessed.

RevDate: 2025-04-26
CmpDate: 2025-04-26

Li XG, Zhu GS, Cao PJ, et al (2025)

Genome-wide CRISPR-Cas9 screening identifies ITGA8 responsible for abivertinib sensitivity in lung adenocarcinoma.

Acta pharmacologica Sinica, 46(5):1419-1432.

The emergence of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) has improved the prognosis for lung cancer patients with EGFR-driven mutations. However, acquired resistance to EGFR-TKIs poses a significant challenge to the treatment. Overcoming the resistance has primarily focused on developing next-generation targeted therapies based on the molecular mechanisms of resistance or inhibiting the activation of bypass pathways to suppress or reverse the resistance. In this study we developed a novel approach by using CRISPR-Cas9 whole-genome library screening to identify the genes that enhance the sensitivity of lung adenocarcinoma cells to EGFR-TKIs. Through this screening, we revealed integrin subunit alpha 8 (ITGA8) as the key gene that enhanced sensitivity to abivertinib in lung adenocarcinoma. Notably, ITGA8 expression was significantly downregulated in lung adenocarcinoma tissues compared to adjacent normal tissues. Bioinformatics analyses revealed that ITGA8 was positively correlated with the sensitivity of lung adenocarcinoma to abivertinib. We showed that knockdown of ITGA8 significantly enhanced the proliferation, migration and invasion of H1975 cells. Conversely, overexpression of ITGA8 reduced the proliferation migration and invasion of H1975/ABIR cells. Furthermore, we demonstrated that ITGA8 sensitized lung adenocarcinoma cells to EGFR-TKIs by attenuating the downstream FAK/SRC/AKT/MAPK signaling pathway. In H1975 cell xenograft mouse models, knockdown of ITGA8 significantly increased tumor growth and reduced the sensitivity to abivertinib, whereas overexpression of ITGA8 markedly suppressed tumor proliferation and enhanced sensitivity to the drug. This study demonstrates that ITGA8 inhibits the proliferation, invasion and migration of lung adenocarcinoma cells, enhances the sensitivity to EGFR-TKIs, improves treatment efficacy, and delays the progression of acquired resistance. Thus, ITGA8 presents a potential therapeutic candidate for addressing acquired resistance to EGFR-TKIs from a novel perspective.

RevDate: 2025-04-26
CmpDate: 2025-01-22

Hołubowicz R, Du SW, Felgner J, et al (2025)

Safer and efficient base editing and prime editing via ribonucleoproteins delivered through optimized lipid-nanoparticle formulations.

Nature biomedical engineering, 9(1):57-78.

Delivering ribonucleoproteins (RNPs) for in vivo genome editing is safer than using viruses encoding for Cas9 and its respective guide RNA. However, transient RNP activity does not typically lead to optimal editing outcomes. Here we show that the efficiency of delivering RNPs can be enhanced by cell-penetrating peptides (covalently fused to the protein or as excipients) and that lipid nanoparticles (LNPs) encapsulating RNPs can be optimized for enhanced RNP stability, delivery efficiency and editing potency. Specifically, after screening for suitable ionizable cationic lipids and by optimizing the concentration of the synthetic lipid DMG-PEG 2000, we show that the encapsulation, via microfluidic mixing, of adenine base editor and prime editor RNPs within LNPs using the ionizable lipid SM102 can result in in vivo editing-efficiency enhancements larger than 300-fold (with respect to the delivery of the naked RNP) without detectable off-target edits. We believe that chemically defined LNP formulations optimized for RNP-encapsulation stability and delivery efficiency will lead to safer genome editing.

RevDate: 2025-04-26
CmpDate: 2025-04-26

Rosenberg-Mogilevsky A, Siegfried Z, R Karni (2025)

Generation of tumor neoantigens by RNA splicing perturbation.

Trends in cancer, 11(1):12-24.

Immunotherapy has revolutionized cancer treatment, but the limited availability of tumor-specific neoantigens still remains a challenge. The potential of alternative mRNA splicing-derived neoantigens as a source of new immunotherapy targets has gained significant attention. Tumors exhibit unique splicing changes and splicing factor mutations which are prevalent in various cancers and play a crucial role in neoantigen production. We present advances in splicing modulation approaches, including small-molecule drugs, decoy and splice-switching antisense oligonucleotides (SSOs), CRISPR, small interfering RNAs (siRNAs), and nonsense-mediated RNA decay (NMD) inhibition, that can be adapted to enhance antitumor immune responses. Finally, we explore the clinical implications of these approaches, highlighting their potential to transform cancer immunotherapy and broaden its efficacy.

RevDate: 2025-04-26
CmpDate: 2025-04-26

Michicich M, Traylor Z, McCoy C, et al (2025)

A W1282X cystic fibrosis mouse allows the study of pharmacological and gene-editing therapeutics to restore CFTR function.

Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, 24(1):164-174.

BACKGROUND: People with cystic fibrosis carrying two nonsense alleles lack CFTR-specific treatment. Growing evidence supports the hypothesis that nonsense mutation identity affects therapeutic response, calling for mutation-specific CF models. We describe a novel W1282X mouse model and compare it to an existing G542X mouse.

METHODS: The W1282X mouse was created using CRISPR/Cas9 to edit mouse Cftr. In this model, Cftr transcription was assessed using qRT-PCR and CFTR function was measured in the airway by nasal potential difference and in the intestine by short circuit current. Growth, survival, and intestinal motility were examined as well. Correction of W1282X CFTR was assessed pharmacologically and by gene-editing using a forskolin-induced swelling (FIS) assay in small intestine-derived organoids.

RESULTS: Homozygous W1282X mice demonstrate decreased Cftr mRNA, little to no CFTR function, and reduced survival, growth, and intestinal motility. W1282X organoids treated with various combinations of pharmacologic correctors display a significantly different amount of CFTR function than that of organoids from G542X mice. Successful gene editing of W1282X to wildtype sequence in intestinal organoids was achieved leading to restoration of CFTR function.

CONCLUSIONS: The W1282X mouse model recapitulates common human manifestations of CF similar to other CFTR null mice. Despite the similarities between the congenic W1282X and G542X models, they differ meaningfully in their response to identical pharmacological treatments. This heterogeneity highlights the importance of studying therapeutics across genotypes.

RevDate: 2025-04-24
CmpDate: 2025-04-25

Mukherjee A, Samanta S, Das S, et al (2025)

Leveraging CRISPR-Cas-Enhanced Isothermal Amplification Tools for Quick Identification of Pathogens Causing Livestock Diseases.

Current microbiology, 82(6):260.

Prompt and accurate diagnosis of infectious pathogens of livestock origin is of utmost importance for epidemiological surveillance and effective therapeutic strategy formulation. Among various methods, nucleic acid-based detection of pathogens is the most sensitive and specific; but the majority of these assays need expensive equipment and skilled workers. Due to the rapid advancement of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based nucleic acid detection methods, these are now being widely used for pathogen detection. CRISPR-Cas is a bacterial counterpart of "adaptive immunity", generally used for editing genome. Many CRISPR systems have been modified for nucleic acid detection due to their excellent selectivity in detecting DNA and RNA sequences. The combination of CRISPR with suitable isothermal amplification technologies has made it more sensitive, specific, versatile, and reproducible for the detection of pathogen nucleic acids at the point of care. Amplification of pathogen nucleic acid by isothermal amplification followed by CRISPR-Cas-based detection has several advantages, including short sample-to-answer times and no requirement for laboratory set-up. They are also significantly less expensive than the existing nucleic acid detection methods. This review focuses on the recent trends in the use of this precision diagnostic method for diagnosis of a wide range of animal pathogens with or without zoonotic potential, particularly various isothermal amplification strategies, and visualization methods for sensing bacteria, viruses, and parasites of veterinary and public health importance.

RevDate: 2025-04-24

Shi H, Al-Sayyad N, Wasko KM, et al (2025)

Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.

Molecular cell pii:S1097-2765(25)00301-6 [Epub ahead of print].

RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.

RevDate: 2025-04-24

Zheng M, Bao N, Wang Z, et al (2025)

Alternative splicing in autism spectrum disorder: Recent insights from mechanisms to therapy.

Asian journal of psychiatry, 108:104501 pii:S1876-2018(25)00144-3 [Epub ahead of print].

Alternative splicing (AS) is a vital and highly dynamic RNA regulatory mechanism that allows a single gene to generate multiple mRNA and protein isoforms. Dysregulation of AS has been identified as a key contributor to the pathogenesis of autism spectrum disorders (ASD). A comprehensive understanding of aberrant splicing mechanisms and their functional consequences in ASD can help uncover the molecular basis of the disorder and facilitate the development of therapeutic strategies. This review focuses on the major aberrant splicing events and key splicing regulators associated with ASD, highlighting their roles in linking defective splicing to ASD pathogenesis. In addition, a discussion of how emerging technologies, such as long-read sequencing, single-cell sequencing, spatial transcriptomics and CRISPR-Cas systems are offering novel insights into the role and mechanisms of AS in ASD is presented. Finally, the RNA splicing-based therapeutic strategies are evaluated, emphasizing their potential to address unmet clinical needs in ASD treatment.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Hayes VM, Zhang JT, Katz MA, et al (2025)

RNA-mediated CRISPR-Cas13 inhibition through crRNA structural mimicry.

Science (New York, N.Y.), 388(6745):387-391.

To circumvent CRISPR-Cas immunity, phages express anti-CRISPR factors that inhibit the expression or activities of Cas proteins. Whereas most anti-CRISPRs described to date are proteins, recently described small RNAs called RNA anti-CRISPRs (rAcrs) have sequence homology to CRISPR RNAs (crRNAs) and displace them from cognate Cas nucleases. In this work, we report the discovery of rAcrVIA1-a plasmid-encoded small RNA that inhibits the RNA-targeting CRISPR-Cas13 system in its natural host, Listeria seeligeri. We solved the cryo-electron microscopy structure of the Cas13-rAcr complex, which revealed that rAcrVIA1 adopts a fold nearly identical to crRNA despite sharing negligible sequence similarity. Collectively, our findings expand the diversity of rAcrs and reveal an example of immune antagonism through RNA structural mimicry.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Akiyama C, Sakata S, F Ono (2025)

Normal locomotion in zebrafish lacking the sodium channel NaV1.4 suggests that the need for muscle action potentials is not universal.

PLoS biology, 23(4):e3003137.

Extensive studies over decades have firmly established the concept that action potentials (APs) in muscles are indispensable for muscle contraction. To re-examine the significance of APs, we generated zebrafish lacking APs by editing the scn4aa and scn4ab genes, which together encode NaV1.4 (NaVDKO), using the CRISPR-Cas9 system. Surprisingly, the escape response of NaVDKOs to tactile stimuli, both in the embryonic and adult stages, was indistinguishable from that of wild-type (WT) fish. Ca2+ imaging using the calcium indicator protein GCaMP revealed that myofibers isolated from WT fish could be excited by the application of acetylcholine (ACh), even in the presence of tetrodotoxin (TTX) indicating that NaVs are dispensable for skeletal muscle contraction in zebrafish. Mathematical simulations showed that the end-plate potential was able to elicit a change in membrane potential large enough to activate the dihydropyridine receptors of the entire muscle fiber owing to the small fiber size and the disseminated distribution of neuromuscular synapses in both adults and embryos. Our data demonstrate that NaVs are not essential for muscle contraction in zebrafish and that the physiological significance of NaV1.4 in muscle is not uniform across vertebrates.

RevDate: 2025-04-25
CmpDate: 2025-04-24

Caras I, Ionescu IE, Pantazica AM, et al (2025)

Humanized mouse model reveals the immunogenicity of Hepatitis B Virus vaccine candidates produced in CRISPR/Cas9-edited Nicotiana benthamiana.

Frontiers in immunology, 16:1479689.

INTRODUCTION: Hepatitis B Virus (HBV) infection is still an ongoing public health issue worldwide. The most efficient tool in preventing HBV infection remains vaccination and significant efforts have been made in the last decade to improve current HBV vaccines. Owing to the strict HBV tropism for the human liver, developing animal models for preclinical screening of vaccine candidates is extremely challenging. To date, there are only a few reports regarding the use of humanized mouse models for the evaluation of the immunogenic properties of viral antigens.

METHODS: Previously we showed that a Nicotiana benthamiana-produced HBV-S/preS1[16-42] antigen elicited strong HBV-specific immune responses in BALB/c mice. In the current study, we used immunodeficient NOD.Cg-Prkdc[scid] Il2rg[tm1Wjl]/SzJ (NSG) mice as recipients of human peripheral blood mononuclear cells (hPBMCs), to evaluate the immunogenicity of the recently developed chimeric HBV immunogen produced in CRISPR/Cas9-edited N. benthamiana, under more "humanized" conditions.

RESULTS: Analysis of the immune response in NSG mice immunized with the chimeric antigen demonstrated induction of virus infection-neutralizing antibodies, indicating activation of antigen-specific B cells.

DISCUSSION: The ability of hPBMCs-engrafted NSG mice to mount specific humoral immune responses after immunization with viral antigens supports this animal model as a promising tool for pre-clinical evaluation of human vaccine antigens.

RevDate: 2025-04-25

Lin XL, Zhou YM, Meng K, et al (2025)

CRISPR/Cas-mediated mRNA knockdown in the embryos of Xenopus tropicalis.

Cell & bioscience, 15(1):52.

The Xenopus tropicalis (Western clawed frog) is an important amphibian model for genetics, developmental and regenerative biology, due to its diploid genetic background and short generation time. CRISPR-Cas13 and CRISPR interference (CRISPRi) systems have recently been employed to suppress mRNA expression in many organisms such as yeast, plants, and mammalian cells. However, no systematic study of these two systems has been carried out in Xenopus tropicalis. Here, we show that CRISPRi rather than CRISPR-Cas13 is an effective and suitable approach to suppress specific mRNA transcription in Xenopus tropicalis embryos. We demonstrated that CRISPRi composed of dCas9 and KRAB-MeCP2 (dCas9-KM) can efficiently target exogenous and endogenous transcripts in Xenopus tropicalis embryos. Moreover, our data suggest that the new KRAB domain from ZIM3 protein (ZIM3-KRAB, ZIM3K) alone has a comparable transcript targeting capacity in Xenopus tropicalis embryos to the traditional fusion repressor KRAB-MeCP2 in which the KRAB domain from KOX1 protein. In conclusion, our results demonstrate that CRISPRi rather than CRISPR-Cas13 is an efficient knockdown platform to explore specific gene function in Xenopus tropicalis embryos.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Chen Z, Pilehvar E, Sadeghi H, et al (2025)

Precision Reimagined: CRISPR and Multiomics Transform Systemic Lupus Erythematosus Diagnosis and Therapy.

International journal of rheumatic diseases, 28(4):e70189.

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with diverse clinical manifestations and unpredictable progression, posing significant challenges to accurate diagnosis and effective treatment. Traditional biomarkers and treatments often fail to address the disease's molecular and clinical heterogeneity. Recent advancements in CRISPR gene-editing technology and multiomics approaches offer transformative opportunities for personalized SLE care by unraveling its underlying molecular complexity and enabling precise therapeutic interventions. CRISPR technology allows targeted editing of SLE-associated genetic mutations, addressing disease drivers directly, while multiomics-including genomics, transcriptomics, and proteomics-provides insights into dysregulated immune networks, identifying biomarkers and therapeutic targets. Integrating these approaches can refine patient stratification and enhance the precision of treatments. Artificial intelligence (AI) complements these technologies by synthesizing high-dimensional data, enabling personalized treatment plans, predicting disease trajectories, and optimizing therapeutic strategies. However, the integration of CRISPR and multiomics in clinical settings raises challenges, including technical limitations, ethical concerns, and economic barriers. Emerging clinical trials and case studies demonstrate the potential of these innovations to personalize care and improve outcomes. Nonetheless, the transition from experimental research to routine clinical application requires robust regulatory frameworks and strategies to address these challenges. This review aims to explore the potential of CRISPR and multiomics technologies to revolutionize SLE diagnosis and therapy, emphasizing their integration with AI to advance personalized care. By addressing existing barriers, the review envisions a future where precision medicine transforms SLE management, paving the way for individualized, patient-centered autoimmune therapy.

RevDate: 2025-04-25
CmpDate: 2025-04-24

Liu Y, Yao F, Zou J, et al (2025)

RPAD locus controls prostrate growth habit in Oryza nivara.

The plant genome, 18(2):e70032.

The development of ideal plant architecture is crucial for optimizing grain yield in crop breeding. The transition from prostrate growth habit in wild rice to erect growth habit in cultivated rice is one of the important events during rice domestication. Here, we identified a yield-related quantitative trait locus (QTL) cluster on the short arm of chromosome 7 using Teqing/W2014 (Oryza nivara) derived BC3F6 population. The introgression line TIL81 containing this QTL cluster exhibited significantly larger tiller angle, increased tiller numbers, and prostrate growth habit compared to the recipient parent Teqing. Using a segregating F2 population derived from a cross between TIL81 and Teqing, this yield-related QTL cluster was mapped to a similar position as the known rice plant architecture domestication (RPAD) locus controlling rice plant architecture domestication. CRISPR/Cas9-mediated genome (where CRISPR is clustered regularly interspaced short palindromic repeats) editing of four zinc finger transcription factors (OnZnF1, OnZnF6, OnZnF8, and OnZnF9) within the RPAD locus demonstrated their collective involvement in regulating plant architecture and yield-related traits. Notably, the knockout lines harboring all four zinc finger gene mutations exhibited plant architecture traits and grain yield per plant comparable to the control Teqing. These findings demonstrated that RPAD locus in O. nivara functions in prostrate growth habit and provided new insights into the molecular mechanism of plant architecture during rice domestication.

RevDate: 2025-04-23

Zhang X, Ma D, F Liu (2025)

CRISPR Technology and Its Emerging Applications.

Genomics, proteomics & bioinformatics pii:8118834 [Epub ahead of print].

The discovery and iteration of clustered regularly interspaced short palindromic repeats (CRISPR) systems have revolutionized genome editing due to their remarkable efficiency and easy programmability, enabling precise manipulation of genomic elements. Owing to these unique advantages, CRISPR technology has the transformative potential to elucidate biological mechanisms and clinical treatments. This review provides a comprehensive overview of the development and applications of CRISPR technology. After describing the three primary CRISPR-Cas systems-CRISPR-associated protein 9 (Cas9) and Cas12a targeting DNA, and Cas13 targeting RNA-which serve as the cornerstone for technological advancements, we describe a series of novel CRISPR-Cas systems that offer new avenues for research, and then explore the applications of CRISPR technology in large-scale genetic screening, lineage tracing, genetic diagnosis, and gene therapy. As this technology evolves, it holds significant promise for studying gene functions and treating human diseases in the near future.

RevDate: 2025-04-23

Biber J, Gandor C, Becirovic E, et al (2025)

Retina-directed gene therapy: Achievements and remaining challenges.

Pharmacology & therapeutics pii:S0163-7258(25)00074-9 [Epub ahead of print].

Gene therapy is an innovative medical approach that offers new treatment options for congenital and acquired diseases by transferring, correcting, inactivating or regulating genes to supplement, replace or modify a gene function. The approval of voretigene neparvovec (Luxturna), a gene therapy for RPE65-associated retinopathy, has marked a milestone for the field of retinal gene therapy, but has also helped to accelerate the development of gene therapies for genetic diseases affecting other organs. Voretigene neparvovec is a vector based on adeno-associated virus (AAV) that delivers a functional copy of RPE65 to supplement the missing function of this gene. The AAV-based gene delivery has proven to be versatile and safe for the transfer of genetic material to retinal cells. However, challenges remain in treating additional inherited as well as acquired retinopathies with this technology. Despite the high level of activity in this field, no other AAV gene therapy for retinal diseases has been approved since voretigene neparvovec. Ongoing research focuses on overcoming the current restraints through innovative strategies like AAV capsid engineering, dual-AAV vector systems, or CRISPR/Cas-mediated genome editing. Additionally, AAV gene therapy is being explored for the treatment of complex acquired diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) by targeting molecules involved in the pathobiology of the degenerative processes. This review outlines the current state of retinal gene therapy, highlighting ongoing challenges and future directions.

RevDate: 2025-04-25
CmpDate: 2025-04-25

Fidelito G, Todorovski I, Cluse L, et al (2025)

Lipid-metabolism-focused CRISPR screens identify enzymes of the mevalonate pathway as essential for prostate cancer growth.

Cell reports, 44(4):115470.

Dysregulated lipid metabolism plays an important role in prostate cancer, although the understanding of the essential regulatory processes in tumorigenesis is incomplete. We employ a CRISPR-Cas9 screen using a custom human lipid metabolism knockout library to identify essential genes for prostate cancer survival. Screening in three prostate cancer cell lines reveals 63 shared dependencies, with enrichment in terpenoid backbone synthesis and N-glycan biosynthesis. Independent knockout of key genes of the mevalonate pathway reduces cell proliferation. Further investigation focuses on NUS1, a subunit of cis-prenyltransferase required for dolichol synthesis. NUS1 knockout decreases tumor growth in vivo and viability in patient-derived xenograft (PDX)-derived organoids. Mechanistic studies reveal that loss of NUS1 promotes oxidative stress, lipid peroxidation and ferroptosis sensitivity, endoplasmic reticulum (ER) stress, and G1 cell-cycle arrest, and it dampens androgen receptor (AR) signaling, collectively leading to growth arrest. This study highlights the critical role of the mevalonate-dolichol-N-glycan biosynthesis pathway, particularly NUS1, in prostate cancer survival and growth.

RevDate: 2025-04-23
CmpDate: 2025-04-24

Brumage L, Best S, Hippe DS, et al (2025)

In vivo functional screens reveal KEAP1 loss as a driver of chemoresistance in small cell lung cancer.

Science advances, 11(17):eadq7084.

Exquisitely chemosensitive initially, small cell lung cancer (SCLC) exhibits dismal outcomes owing to rapid transition to chemoresistance. Elucidating the genetic underpinnings has been challenging owing to limitations with cellular models. As SCLC patient-derived xenograft (PDX) models mimic therapeutic responses, we perform genetic screens in chemosensitive PDX models to identify drivers of chemoresistance. cDNA overexpression screens identify MYC, MYCN, and MYCL, while CRISPR deletion screens identify KEAP1 loss as driving chemoresistance. Deletion of KEAP1 switched a chemosensitive SCLC PDX model to become chemoresistant and resulted in sensitivity to inhibition of glutamine metabolism. Data from the IMpower133 clinical trial revealed ~6% of patients with extensive-stage SCLC exhibit KEAP1 genetic alterations, with activation of a KEAP1/NRF2 transcriptional signature associated with reduced survival upon chemotherapy treatment. While roles for KEAP1/NRF2 have been unappreciated in SCLC, our genetic screens revealed KEAP1 loss as a driver of chemoresistance, while patient genomic analyses demonstrate clinical importance.

RevDate: 2025-04-23
CmpDate: 2025-04-23

Zeng Y, Tan X, Xiao P, et al (2025)

Natronobacterium gregoryi Argonaute inhibits class 1 integron integrase-mediated excision and integration.

Nucleic acids research, 53(8):.

Argonaute (Ago) proteins, ubiquitous in all domains of life, serve as key components in defense against foreign nucleic acids. While eukaryotic Agos (eAgos) are well characterized for guide RNA-mediated RNA targeting, prokaryotic Agos (pAgos) exhibit diverse functions, particularly in protecting bacteria from invasive DNA. The previous study identified Class 1 integron integrase (IntI-1), a tyrosine site-specific recombinase involved in horizontal transfer of antibiotic resistance genes, as a potential interaction partner of Natronobacterium gregoryi Argonaute (NgAgo), a member of pAgos. Here, we demonstrated that this interaction was direct, depended on the PIWI domain, and was independent of the catalytic activity of NgAgo. Notably, no interaction occurred between NgAgo and Cre (another tyrosine site-specific recombinase), highlighting the specificity of NgAgo-IntI-1 interaction. Furthermore, NgAgo could inhibit binding of IntI-1 to its target DNA, and then impede IntI-1-mediated integration and excision. Consistent with the above finding, few pAgos could be found in prokaryotic genomes containing IntI, whereas IntI showed significant co-occurrence with another bacterial defense system, CRISPR-Cas. In summary, our study elucidated a novel defense mechanism of pAgos through interaction with IntI-1 for inhibiting IntI-1-mediated gene excision/integration process.

RevDate: 2025-04-23

Han X, Deng Z, Liu H, et al (2025)

Current Advancement and Future Prospects in Simplified Transformation-Based Plant Genome Editing.

Plants (Basel, Switzerland), 14(6): pii:plants14060889.

Recent years have witnessed remarkable progress in plant biology, driven largely by the rapid evolution of CRISPR/Cas-based genome editing (GE) technologies. These tools, including versatile CRISPR/Cas systems and their derivatives, such as base editors and prime editors, have significantly enhanced the universality, efficiency, and convenience of plant functional genomics, genetics, and molecular breeding. However, traditional genetic transformation methods are essential for obtaining GE plants. These methods depend on tissue culture procedures, which are time-consuming, labor-intensive, genotype-dependent, and challenging to regenerate. Here, we systematically outline current advancements in simplifying plant GE, focusing on the optimization of tissue culture process through developmental regulators, the development of in planta transformation methods, and the establishment of nanomaterial- and viral vector-based delivery platforms. We also discuss critical challenges and future directions for achieving genotype-independent, tissue culture-free plant GE.

RevDate: 2025-04-23

Zueva AS, Shevchenko AI, Medvedev SP, et al (2025)

Isogenic induced pluripotent stem cell line ICGi036-A-1 from a patient with familial hypercholesterolaemia, derived by correcting a pathogenic variant of the gene LDLR c.530C>T.

Vavilovskii zhurnal genetiki i selektsii, 29(2):189-199.

Familial hypercholesterolaemia is a common monogenic disorder characterized by high plasma cholesterol levels leading to chronic cardiovascular disease with high risk and often early manifestation due to atherosclerotic lesions of the blood vessels. The atherosclerotic lesions in familial hypercholesterolaemia are mainly caused by pathogenic variants of the low-density lipoprotein receptor (LDLR) gene, which plays an important role in cholesterol metabolism. Normally, cholesterol-laden low-density lipoproteins bind to the LDLR receptor on the surface of liver cells to be removed from the bloodstream by internalisation with hepatocytes. In familial hypercholesterolaemia, the function of the receptor is impaired and the uptake of low-density lipoproteins is significantly reduced. As a result, cholesterol accumulates in the subendothelial space on the inner wall of blood vessels, triggering atherogenesis, the formation of atherosclerotic plaques. At present, there are no effective and universal approaches to the diagnosis and treatment of familial hypercholesterolaemia. A relevant approach to study the molecular genetic mechanisms of the disease and to obtain systems for screening chemical compounds as potential drugs is the generation of cellular models based on patient-specific induced pluripotent stem cells. The aim of our work was to derive an isogenic genetically modified induced pluripotent stem cell line by correcting the pathogenic allelic variant c.530C of the LDLR gene in the original iPSC previously obtained from a compound heterozygote patient with familial hypercholesterolaemia. The resulting isogenic iPSC line differs from the original by only one corrected nucleotide substitution, allowing us to study the direct effect of this pathogenic genetic variant on physiological changes in relevant differentiated cells. CRISPR/Cas-mediated base editing was used to correct the single nucleotide substitution. The resulting genetically modified iPSC line has pluripotency traits, a normal karyotype, a set of short tandem repeats identical to that in the original line and can be used to obtain differentiated derivatives necessary for the elaboration of relevant cell models.

RevDate: 2025-04-23
CmpDate: 2025-04-23

Yahata T, Toujima S, Sasaki I, et al (2025)

Adeno-associated virus-clustered regularly interspaced short palindromic repeats/cas9‑mediated ovarian cancer treatment targeting PD-L1.

BMC cancer, 25(1):749.

The response rate of antibody therapy targeting immune checkpoint molecules in ovarian cancer is insufficient. This study aimed to develop a novel gene immunotherapy model targeting programmed death ligand 1 (PD-L1) in vivo in ovarian cancer using adeno-associated virus (AAV)-clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and investigate its efficacy. In vitro, we produced PD-L1-AAV particles to knock out PD-L1. PD-L1-AAV particles were transduced into the murine ovarian cancer cell line ID8. PD-L1 expression at the cellular level was significantly decreased following treatment with PD-L1-AAV particles compared with control-AAV particles. In the peritoneal dissemination model, the survival time was significantly longer in the PD-L1-AAV particles intraperitoneally injected group than that in the control group. Furthermore, intratumoral lymphocyte recruitment was analyzed by immunohistochemistry, and the number of intratumoral CD4[+] and CD8[+] T cells was significantly higher, whereas that of Foxp3[+] Treg cells was significantly lower in the PD-L1-AAV particles injected group than in the control group. No severe adverse events in normal organs, such as the lungs, spleen, liver, and kidney, were observed. These results suggest that PD-L1-targeted therapy by genome editing using AAV-CRISPR/Cas9 is a novel gene-immune therapeutic strategy for ovarian cancer.

RevDate: 2025-04-24
CmpDate: 2025-04-23

Pang KL, Li P, Yao XR, et al (2025)

Deciphering a proliferation-essential gene signature based on CRISPR-Cas9 screening to predict prognosis and characterize the immune microenvironment in HNSCC.

BMC cancer, 25(1):756.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive malignancy with a poor prognosis. Identifying reliable prognostic biomarkers and therapeutic targets is crucial for improving patient outcomes. This study aimed to systematically identify proliferation-essential genes (PEGs) associated with HNSCC prognosis using CRISPR-Cas9 screening data.

METHODS: CRISPR-Cas9 screening data from the DepMap database were used to identify PEGs in HNSCC cells. A prognostic PEGs signature was constructed using univariate Cox regression, least absolute shrinkage and selection operator (LASSO) Cox regression, and multivariate Cox regression analyses. The predictive accuracy of the signature was validated in internal and external datasets. Weighted gene co-expression network analysis (WGCNA), gene set enrichment analysis (GSEA), and immune infiltration analysis were used to investigate the underlying mechanism between high and low-risk patients. Random forest analysis and functional experiments were conducted to investigate the role of key proliferation essential genes in HNSCC progression.

RESULTS: A total of 1511 PEGs were identified. A seven-gene prognostic PEGs signature (MRPL33, NAT10, PSMC1, PSMD11, RPN2, TAF7, and ZNF335) was developed and validated, demonstrating robust prognostic performance in stratifying HNSCC patients by survival risk. WGCNA and GSEA analyses revealed a marked downregulation of immune-related pathways in high-risk patients. Immune infiltration analysis validated those high-risk patients had reduced immune scores, stromal scores, and ESTIMATE scores, as well as decreased infiltration of multiple immune cell types. Among the identified genes, PSMC1 was highlighted as a pivotal regulator of HNSCC proliferation and migration, as confirmed by functional experiments.

CONCLUSIONS: This study identifies a novel PEGs signature that effectively predicts HNSCC prognosis and stratifies patients by survival risk. PSMC1 was identified as a key gene promoting malignant progression, offering potential as a therapeutic target for HNSCC.

RevDate: 2025-04-23
CmpDate: 2025-04-23

Huang X, Li R, Xu J, et al (2025)

Integrated multi-omics uncover viruses, active fermenting microbes and their metabolic profiles in the Daqu microbiome.

Food research international (Ottawa, Ont.), 208:116061.

The coexistence and coevolution of viruses and fermenting microbes have a significant impact on the structure and function of microbial communities. Although the presence of viruses in Daqu, the fermentation starter for Chinese Baijiu, has been documented, their specific effects on the community composition and metabolic functions of low, medium, and high-temperature Daqu remain unclear. In this study, we employed multi-omics technology to explore the distribution of viruses and active bacteria and fungi in various Daqu and their potential metabolic roles. Viral metagenomic sequencing showed a predominance of Parvoviridae in High-Temperature Daqu (HTQ), while Genomoviridae were dominant in Medium-Temperature Daqu (MTQ) and Low- Temperature Daqu (LTQ). Phages belonging to the Siphoviridae, Podoviridae, Herelleviridae, and Myoviridae families showed significantly different abundances across three Daqu groups. Metatranscriptomic analysis showed that fungal communities were most active in LTQ, whereas bacterial communities were dominant in MTQ and HTQ. By employing the CRISPR-Cas spacer, a higher predicted number of phage-host linkages was identified in LTQ, particularly with hosts including Lactobacillus, Staphylococcus, Acinetobacter, Enterobacter, and Bacillus. Correlation analysis showed that bacteria like Acinetobacter, Lactobacillus, and Streptococcus exhibited the strongest associations with metabolites, particularly amino acids and organic acids. The potential phage-induced metabolic differences in the three Daqu groups were mainly linked to pathways involved in the metabolism of amino acids, sugars, and organic acids. Overall, our study elucidates the impact of viruses on shaping microbial composition and influencing metabolic functions in Daqu. These results improve our comprehension of viruses and microbes in Daqu microbial communities and provide valuable insights for enhancing quality control in Daqu production.

RevDate: 2025-04-23
CmpDate: 2025-04-23

Zhu Z, Li X, Ding L, et al (2025)

Exploring the effect of activator topology on CRISPR-Cas12a trans-cleavage activity.

Nucleic acids research, 53(8):.

The CRISPR-Cas12a system is widely used in nucleic acid detection and biosensing due to its high sensitivity, selectivity, and simple design. However, traditional CRISPR-Cas12a sensors, which rely on linear activators, face challenges such as limited operability and low stability. This study explored the impact of three different activator topologies-linear, planar, and steric-on the trans-cleavage activity of Cas12a. We developed a Cas12a-based switch using a planar activator, which demonstrated superior operability and maintained higher activity compared to linear activators. Using this planar activator, we achieved highly sensitive detection of hypochlorous acid, with a detection limit as low as 88 nM, outperforming chemical probe-based methods. The introduction of topological activators will open new avenues for the development of CRISPR-Cas12a-based biosensors, offering broad potential for diverse applications.

RevDate: 2025-04-22

Wood TWP, Henriques WS, Cullen HB, et al (2025)

The retrotransposon-derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.

Nature aging [Epub ahead of print].

Almost half of the human genome consists of retrotransposons-'parasitic' sequences that insert themselves into the host genome via an RNA intermediate. Although most of these sequences are silenced or mutationally deactivated, they can present opportunities for evolutionary innovation: mutation of a deteriorating retrotransposon can result in a gene that provides a selective advantage to the host in a process termed 'domestication'[1-3]. The PNMA family of gag-like capsid genes was domesticated from an ancient vertebrate retrotransposon of the Metaviridae clade at least 100 million years ago[4,5]. PNMA1 and PNMA4 are positively regulated by the master germ cell transcription factors MYBL1 and STRA8, and their transcripts are bound by the translational regulator DAZL during gametogenesis[6]. This developmental regulation of PNMA1 and PNMA4 expression in gonadal tissue suggested to us that they might serve a reproductive function. Through the analysis of donated human ovaries, genome-wide association studies (GWASs) and mouse models, we found that PNMA1 and PNMA4 are necessary for the maintenance of a normal reproductive lifespan. These proteins self-assemble into capsid-like structures that exit human cells, and we observed large PNMA4 particles in mouse male gonadal tissue that contain RNA and are consistent with capsid formation.

RevDate: 2025-04-24

Weiss T, Kamalu M, Shi H, et al (2025)

Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis.

Nature plants [Epub ahead of print].

Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation[1]. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Li Z, Wang N, Wang H, et al (2025)

Single-cell transcriptomics reveals the mechanisms of lung injury induced by galt gene editing in mouse.

Biochemical and biophysical research communications, 763:151780.

Galactosemia, caused by mutations in the GALT gene, leads to multi-organ damage. This study investigates the impact of Galt c.847 + 1G > T mutation on lung tissue using single-cell transcriptomics. We employed CRISPR/Cas9 to generate a Galt gene-edited mouse model with the Galt c. 847 + 1G > T mutation and assessed Galt expression through PCR and Western blotting. Histopathological analysis revealed significant structural lung changes, including alveolar congestion and inflammation. Single-cell RNA sequencing demonstrated a marked reduction in immune cells (NK, T, macrophages, B cells) and an increase in alveolar type II cells, vascular endothelial cells, and myofibroblasts in the GAL mouse. The increased abundance of alveolar type II cells indicated impaired differentiation and repair. Metabolic analysis revealed significant abnormalities linked to Galt c.847 + 1G > T mutation, with disruptions in TGF-β1, FGF, and Mif pathways contributing to cellular dysfunction and exacerbated lung injury. This model provides insights into the molecular mechanisms of lung injury in galactosemia, highlighting significant alterations in lung cell populations and key signaling pathways.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Zhang Y, Lu Z, Yang H, et al (2025)

Generation of stable Cas9-EGFP expressing human induced pluripotent stem cell lines based on SeLection by Essential-gene Exon Knock-in technology.

Stem cell research, 85:103710.

Here, we used SeLection by Essential-gene Exon Knock-in technology to generate the iPSC line with constitutive expression of Cas9-EGFP, while retaining all functions of the essential gene. Cas9-EGFP was inserted into the GAPDH exon9 via the homologous recombination, avoiding Cas9 silencing that often occurs during iPSC differentiation. The edited cell line shows precise knock-in locus with the typical characteristics and pluripotency of iPSCs. Therefore, this iPSC line is valuable for CRISPR screening or related experiments and could be widely used in the CRISPR/Cas9-based gene editing.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Fielden J, Siegner SM, Gallagher DN, et al (2025)

Comprehensive interrogation of synthetic lethality in the DNA damage response.

Nature, 640(8060):1093-1102.

The DNA damage response (DDR) is a multifaceted network of pathways that preserves genome stability[1,2]. Unravelling the complementary interplay between these pathways remains a challenge[3,4]. Here we used CRISPR interference (CRISPRi) screening to comprehensively map the genetic interactions required for survival during normal human cell homeostasis across all core DDR genes. We captured known interactions and discovered myriad new connections that are available online. We defined the molecular mechanism of two of the strongest interactions. First, we found that WDR48 works with USP1 to restrain PCNA degradation in FEN1/LIG1-deficient cells. Second, we found that SMARCAL1 and FANCM directly unwind TA-rich DNA cruciforms, preventing catastrophic chromosome breakage by the ERCC1-ERCC4 complex. Our data yield fundamental insights into genome maintenance, provide a springboard for mechanistic investigations into new connections between DDR factors and pinpoint synthetic vulnerabilities that could be exploited in cancer therapy.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Su M, Zhang HS, Liu H, et al (2025)

Allosteric ribozyme-driven crRNA switch for the amplification-free detection of biomolecules.

Biosensors & bioelectronics, 280:117450.

Currently, CRISPR-mediated biosensors are concentrating on the design of the crRNA or the activator strand to regulate the trans-cleavage activity of Cas12a. Herein, we report an allosteric ribozyme-driven crRNA switch-regulated CRISPR/Cas12a sensor for amplification-free detection of biomolecules. An allosteric ribozyme is meticulously engineered to connect the target recognition sequence with the 5' binding arm of the hammerhead ribozyme, resulting in the formation of a hairpin structure through complementary hybridization. The presence of target induces the conformational change in the allosteric module and disrupts the hairpin structure, restoring multiple-turnover cleavage RNA activity of ribozyme. Then, the activated ribozyme specifically cuts the cleavage site of the substrate-locked crRNA and releases the native crRNA to initiate CRISPR/Cas12a functions for signal reporting. The reported biosensor exhibited high sensitivity and excellent specificity for miR-155 and adenosine triphosphate (ATP) detection, giving the detection limits of 256 fM and 160 nM, respectively. For clinical validation, our proposed strategy can quantify miR-155 expression levels in cells and serum of cancer patients. Furthermore, we also demonstrate that the allosteric ribozyme-driven crRNA switch can be easily compatible with lateral flow assays, realizing visualization and the portable monitoring of target. Hence, the biosensor not only has outstanding potential in point-of-care testing, but also enables the detection of various biomolecules by flexibly substituting target recognition sequences for molecular diagnosis in the clinic.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Wu Z, Xu Y, Zhou W, et al (2025)

Rapid detection of Klebsiella pneumoniae based on one-tube RPA-CRISPR/Cas12a system.

Clinica chimica acta; international journal of clinical chemistry, 573:120281.

Klebsiella pneumoniae (KP) is a prevalent pathogen implicated in both community-acquired and nosocomial infections, often leading to severe clinical outcomes. The conventional methods for KP identification are characterized by intricacy and suboptimal efficiency. In this research, we have engineered a novel One-Tube RPA- CRISPR/Cas12a system, integrating recombinase polymerase amplification (RPA) method with the CRISPR/Cas12a diagnostic platform, to facilitate the detection of K. pneumoniae. To minimize the likelihood of aerosol-based contamination, the RPA components are positioned at the base of the tube, while the CRISPR/Cas12a components are placed at the tube's cap. The systems are combined post-RPA amplification through a brief centrifugation step, ensuring that RPA reactions are conducted independently to produce an adequate amount of target DNA before interaction with the CRISPR/Cas12a system. This method was validated using both fluorescent and lateral flow strip assays, achieving a limit of detection (LOD) of 10[0] copies/μL and 10[1] copies/μL respectively. The specificity for KP detection was found to be 100 %. Furthermore, the system demonstrated a positivity rate of 78 % (18/23) when directly extracting DNA from sputum samples, corroborated by culture and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The simplicity and rapidity of the assay are augmented by a straightforward sample processing without extraction. The complete assay duration from specimen receipt to result is approximately 40 min, significantly reducing the turnaround time (TAT). Collectively, this system presents a streamlined, expeditious, and highly specific diagnostic approach for the detection of Klebsiella pneumoniae strains.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Hu H, Xue H, Dong K, et al (2025)

Strand displacement-enhanced CRISPR-Cas13a system for ultra-specific detection of RNA single nucleotide variation.

Biosensors & bioelectronics, 280:117445.

RNA plays a critical role in biological systems, mediating genetic information transfer and regulating gene expression. However, RNA is susceptible to variations from endogenous and exogenous sources, with potentially profound biological consequences. The CRISPR-Cas13a system has emerged as a promising tool for RNA variation detection due to its cost-effectiveness, sensitivity, and user-friendly nature. Despite this, designing a simple, universal system with high discrimination factor (DF) for single-nucleotide variations remains a challenge. Here, we present the strand displacement-enhanced Cas13a single-nucleotide variation detection assay (SECND), a sensitive, universal, and easy-to-implement method with a high DF for RNA variations. Using SECND, we detected 5 types of single-nucleotide variations, achieving a maximum DF of 1083.2. We validated the assay's effectiveness on miRNA and SARS-CoV-2 genomic RNA simulants, incorporating a 4-way strand displacement mechanism to enhance detection limits to 10 pmol/L and 50 pmol/L, and to identify variations at frequencies as low as 0.01 % and 0.1 %. Additionally, we demonstrated SECND's utility in quantifying single-nucleotide variants of miR-200b and miR-200c in ovarian cancer and retinal glioma cells. This versatile tool not only advances RNA variation detection but also has significant implications for disease research, diagnostics, and viral classification, enhancing our understanding of the CRISPR-Cas13a system and its potential applications.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Hu F, Zhang Y, Yang Y, et al (2025)

A rapid and ultrasensitive RPA-assisted CRISPR-Cas12a/Cas13a nucleic acid diagnostic platform with a smartphone-based portable device.

Biosensors & bioelectronics, 280:117428.

The spread of infectious diseases can be controlled by early identification of the source of infection and timely diagnosis to stop transmission. Real-time fluorescence quantitative polymerase chain reaction (PCR) is the current gold standard for pathogen diagnosis, with high detection sensitivity and accuracy. However, due to the need for specialized equipment, laboratories, and personnel, it is difficult to achieve rapid and immediate diagnosis during large-scale infectious disease outbreaks. Herein, an optimized CRISPR-based nucleic acid detection method was developed that reduces the CRISPR detection time to 15 min while maintaining high sensitivity. By using nucleic acid extraction-free and lyophilization techniques, the 'sample-in-result-out' detection of the two target genes of SARS-CoV-2, the human internal reference gene, and the negative quality control sample can be completed in 20 min, with a sensitivity of 0.5 copies/μL. Additionally, to facilitate the application, a smartphone-based reverse transcription-recombinase polymerase amplification (RT-RPA)-assisted CRISPR-rapid, portable nucleic acid detection device was developed, integrating functions such as heating, centrifugation, mixing, optical detection and result output. Process control, output, and uploading of detection results were conducted through smartphones. The device is not dependent on a power supply and can perform on-site rapid virus detection in resource-limited settings. Real-time uploading of results helps to rapidly implement epidemic prevention and control measures, providing an innovative means of detection, control, and prevention of virus-based infectious diseases. This important work provides a new and effective tool to manage potential future outbreaks of infectious diseases.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Li J, Yin L, Wang C, et al (2025)

Generation of a homozygous ABCA7 knockout cell line (AHMUCNi002-A) in human iPSCs using CRISPR/Cas9.

Stem cell research, 85:103700.

ABCA7, located on chromosome 19, encodes an ATP-binding cassette transporter. Loss-of-function variants of ABCA7 are associated with an increased risk of Alzheimer's disease. To explore the role of ABCA7 deficiency in the pathogenesis of Alzheimer's disease, CRISPR/Cas9 genome-editing technology was utilized to generate a homozygous ABCA7 knockout in human induced pluripotent stem cells (hiPSCs). The resulting ABCA7 knockout cell line exhibited normal pluripotency, a stable karyotype, and the ability to differentiate into all three germ layers.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Zhou H, Liu Y, Chen Q, et al (2025)

Generation of TP53 knock out induced pluripotent stem cell using CRISPR/Cas9.

Stem cell research, 85:103699.

The TP53 gene is an important tumor suppressor gene. Through CRISPR/Cas9 technology, we have established a TP53 gene knockout cell line in iPSCs (SIIBRi001-A). This cell line maintains normal stem cell-like morphology, karyotype, expresses markers of pluripotency, and is capable of generating teratomas in immunodeficient mice. Quantitative analysis of pluripotency gene expression remains normal. This cell line can be utilized for studying the mechanisms underlying tumorigenesis.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Ludwik KA, Opitz R, Jyrch S, et al (2025)

Correction of the Allan-Herndon-Dudley syndrome-causing SLC16A2 mutation G401R in a patient derived hiPSC line.

Stem cell research, 85:103698.

The X-linked Allan-Herndon-Dudley syndrome (AHDS) is a genetic disorder characterized by severe psychomotor impairment, resulting from mutations in the SLC16A2 gene, which encodes the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). Previously, we established a hiPSC line from a patient carrying the SLC16A2:R401G mutation (BIHi045-A). Using CRISPR/Cas9-mediated gene editing, we targeted exon 3 of SLC16A2 and used single-stranded oligodeoxynucleotides as homology-directed repair templates to correct the R401G missense mutation, generating an isogenic control cell line.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Yuan S, Sun R, Shi H, et al (2025)

VDAC2 loss elicits tumour destruction and inflammation for cancer therapy.

Nature, 640(8060):1062-1071.

Tumour cells often evade immune pressure exerted by CD8[+] T cells or immunotherapies through mechanisms that are largely unclear[1,2]. Here, using complementary in vivo and in vitro CRISPR-Cas9 genetic screens to target metabolic factors, we established voltage-dependent anion channel 2 (VDAC2) as an immune signal-dependent checkpoint that curtails interferon-γ (IFNγ)-mediated tumour destruction and inflammatory reprogramming of the tumour microenvironment. Targeting VDAC2 in tumour cells enabled IFNγ-induced cell death and cGAS-STING activation, and markedly improved anti-tumour effects and immunotherapeutic responses. Using a genome-scale genetic interaction screen, we identified BAK as the mediator of VDAC2-deficiency-induced effects. Mechanistically, IFNγ stimulation increased BIM, BID and BAK expression, with VDAC2 deficiency eliciting uncontrolled IFNγ-induced BAK activation and mitochondrial damage. Consequently, mitochondrial DNA was aberrantly released into the cytosol and triggered robust activation of cGAS-STING signalling and type I IFN response. Importantly, co-deletion of STING signalling components dampened the therapeutic effects of VDAC2 depletion in tumour cells, suggesting that targeting VDAC2 integrates CD8[+] T cell- and IFNγ-mediated adaptive immunity with a tumour-intrinsic innate immune-like response. Together, our findings reveal VDAC2 as a dual-action target to overcome tumour immune evasion and establish the importance of coordinately destructing and inflaming tumours to enable efficacious cancer immunotherapy.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Blomme J, Arraiza Ribera J, De Clerck O, et al (2025)

Consolidating Ulva functional genomics: gene editing and new selection systems.

The New phytologist, 246(4):1710-1723.

The green seaweed Ulva compressa is a promising model for functional biology. In addition to historical research on growth and development, -omics data and molecular tools for stable transformation are available. However, more efficient tools are needed to study gene function. Here, we expand the molecular toolkit for Ulva. We screened the survival of Ulva and its mutualistic bacteria on 14 selective agents and established that Blasticidin deaminases (BSD or bsr) can be used as selectable markers to generate stable transgenic lines. We show that Cas9 and Cas12a RNPs are suitable for targeted mutagenesis and can generate genomic deletions of up to 20 kb using the marker gene ADENINE PHOSPHORIBOSYLTRANSFERASE (APT). We demonstrate that the targeted insertion of a selectable marker via homology-directed repair or co-editing with APT is possible for nonmarker genes. We evaluated 31 vector configurations and found that the bicistronic fusion of Cas9 to a resistance marker or the incorporation of introns in Cas9 led to the most mutants. We used this to generate mutants in three nonmarker genes using a co-editing strategy. This expanded molecular toolkit now enables us to reliably make gain- and loss-of-function mutants; additional optimizations will be necessary to allow for vector-based multiplex genome editing in Ulva.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Zhu H, Zhou T, Guan J, et al (2025)

Precise genome editing of Dense and Erect Panicle 1 promotes rice sheath blight resistance and yield production in japonica rice.

Plant biotechnology journal, 23(5):1832-1846.

The primary goals of crop breeding are to enhance yield and improve disease resistance. However, the "trade-off" mechanism, in which signalling pathways for resistance and yield are antagonistically regulated, poses challenges for achieving both simultaneously. Previously, we demonstrated that knock-out mutants of the Dense and Erect Panicle 1 (DEP1) gene can significantly enhance rice resistance to sheath blight (ShB), and we mapped DEP1's association with panicle length. In this study, we discovered that dep1 mutants significantly reduced rice yield. Nonetheless, truncated DEP1 was able to achieve both ShB resistance and yield increase in japonica rice. To further explore the function of truncated DEP1 in promoting yield and ShB resistance, we generated CRISPR/Cas9-mediated genome editing mutants, including a full-length deletion mutant of DEP1, named dep1, and a truncated version, dep1-cys. Upon inoculation with Rhizoctonia solani, the dep1-cys mutant demonstrated stronger ShB resistance than the dep1 mutant. Additionally, dep1-cys increased yield per plant, whereas dep1 reduced it. Compared to the full DEP1 protein, the truncated DEP1 (dep1-cys) demonstrated a decreased interaction affinity with IDD14 and increased affinity with IDD10, which are known to positively and negatively regulate ShB resistance through the activation of PIN1a and ETR2, respectively. The dep1-cys mutant exhibited higher PIN1a and lower ETR2 expression than wild-type plants, suggesting that dep1-cys modulated IDD14 and IDD10 interactions to regulate PIN1a and ETR2, thereby enhancing ShB resistance. Overall, these data indicate that precise genome editing of DEP1 could simultaneously improve both ShB resistance and yield, effectively mitigating trade-off regulation in rice.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Cui X, Yang H, Cai C, et al (2025)

Comparative characterization of human accelerated regions in neurons.

Nature, 640(8060):991-999.

Human accelerated regions (HARs) are conserved genomic loci that have experienced rapid nucleotide substitutions following the divergence from chimpanzees[1,2]. HARs are enriched in candidate regulatory regions near neurodevelopmental genes, suggesting their roles in gene regulation[3]. However, their target genes and functional contributions to human brain development remain largely uncharacterized. Here we elucidate the cis-regulatory functions of HARs in human and chimpanzee induced pluripotent stem (iPS) cell-induced excitatory neurons. Using genomic[4] and chromatin looping information, we prioritized 20 HARs and their chimpanzee orthologues for functional characterization via single-cell CRISPR interference, and demonstrated their species-specific gene regulatory functions. Our findings reveal diverse functional outcomes of HAR-mediated cis-regulation in human neurons, including attenuated NPAS3 expression by altering the binding affinities of multiple transcription factors in HAR202 and maintaining iPS cell pluripotency and neuronal differentiation capacities through the upregulation of PUM2 by 2xHAR.319. Finally, we used prime editing to demonstrate differential enhancer activity caused by several HAR26;2xHAR.178 variants. In particular, we link one variant in HAR26;2xHAR.178 to elevated SOCS2 expression and increased neurite outgrowth in human neurons. Thus, our study sheds new light on the endogenous gene regulatory functions of HARs and their potential contribution to human brain evolution.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Li L, Fu X, Qi X, et al (2025)

Harnessing haploid-inducer mediated genome editing for accelerated maize variety development.

Plant biotechnology journal, 23(5):1604-1614.

The integration of haploid induction and genome editing, termed HI-Edit/IMGE, is a promising tool for generating targeted mutations for crop breeding. However, the technical components and stacking suitable for the maize seed industry have yet to be fully characterised and tested. Here, we developed and assessed three HI-Edit/IMGE maize lines: Edit[Wx], Edit[Sh], and Edit[Wx&Sh], using the haploid inducer CHOI3 and lines engineered using the CRISPR-Cas9 system targeting the Waxy1 (Wx1) and Shrunken2 (Sh2) genes. We meticulously characterised the HI-Edit/IMGE systems, focusing on copy numbers and the mutant alleles mtl and dmp, which facilitate haploid induction. Using B73 and six other parental lines of major commercial varieties as recipients, HI-Edit/IMGE demonstrated maternal haploid induction efficiencies ranging from 8.55% to 20.89% and targeted mutation rates between 0.38% and 1.46%. Comprehensive assessment verified the haploid identification, target gene editing accuracy, genome background integrity, and related agronomic traits. Notably, Edit[Wx&Sh] successfully combined distinct CRISPR-Cas9 systems to induce multiple desired mutations, highlighting the potential of HI-Edit/IMGE in accelerating the integration of edited traits into commercial maize varieties. Our findings underscore the importance of meticulous Cas9 copy number characterisation and highlight potential challenges related to somatic chimerism. We also validated the performance of single-cross haploids derived using the HI-Edit/IMGE process. Our results confirm the industrial applicability of generating targeted mutations through pollination and provide critical insights for further optimising this technology.

RevDate: 2025-04-24
CmpDate: 2025-04-24

van Belle J, Schaart JG, Dechesne AC, et al (2025)

Direct and indirect effects of multiplex genome editing of F5H and FAD2 in oil crop camelina.

Plant biotechnology journal, 23(5):1399-1412.

Mutants with simultaneous germline mutations were obtained in all three F5H genes and all three FAD2 genes (one to eleven mutated alleles) in order to improve the feed value of the seed meal and the fatty acid composition of the seed oil. In mutants with multiple mutated F5H alleles, sinapine in seed meal was reduced by up to 100%, accompanied by a sharp reduction in the S-monolignol content of lignin without causing lodging or stem break. A lower S-lignin monomer content in stems can contribute to improved stem degradability allowing new uses of stems. Mutants in all six FAD2 alleles showed an expected increase in MUFA from 8.7% to 74% and a reduction in PUFA from 53% to 13% in the fatty acids in seed oil. Remarkably, some full FAD2 mutants showed normal growth and seed production and not the dwarfing phenotype reported in previous studies. The relation between germline mutation allele dosage and phenotype was influenced by the still ongoing activity of the CRISPR/Cas9 system, leading to new somatic mutations in the leaves of flowering plants. The correlations between the total mutation frequency (germline plus new somatic mutations) for F5H with sinapine content, and FAD2 with fatty acid composition were higher than the correlations between germline mutation count and phenotypes. This shows the importance of quantifying both the germline mutations and somatic mutations when studying CRISPR/Cas9 effects in situations where the CRISPR/Cas9 system is not yet segregated out.

RevDate: 2025-04-24
CmpDate: 2025-04-24

Xiao L, Qin B, Zhang X, et al (2025)

Precision Control of Cell Type-Specific Behavior via RNA Sensing and Editing.

Small methods, 9(4):e2400952.

In the realms of bioengineering and biopharmaceuticals, there exists a critical demand for advanced genetic tools that can interact with specific cell signaling pathways to accurately identify and target various cell types. This research introduces the innovative CRISPR-ADAReader system, which enables precise manipulation of cell activity through sensing target RNA. Featuring both positive and negative feedback loops, the system allows for tailored regulation across different cell types in response to various internal signals, showcasing exceptional programmability, specificity, and sensitivity. By choosing distinct RNAs as activation signals, the CRISPR-ADAReader efficiently monitors and alters targeted cell behaviors. In a case study focusing on retinoblastoma treatment, the system distinctively initiates positive feedback and self-silencing actions by detecting MCYN and Rb transcripts, thus safeguarding normal retinal pigment epithelial cells while promoting apoptosis in cancer cells. Moreover, the CRISPR-ADAReader demonstrates significant anti-tumor effectiveness in live models, markedly reducing retinoblastoma cell proliferation through the activation of several cancer-suppression pathways, outperforming the capabilities of the ADAR-sensor. Notably, the system also shows an excellent in vivo safety profile. In conclusion, the CRISPR-ADAReader system represents a groundbreaking method for the detection and editing of RNA, offering a potent instrument for the customized and precise governance of cell behavior.

RevDate: 2025-04-22
CmpDate: 2025-04-23

Zhao C, Cao Y, Ibrahim N, et al (2025)

Efficient in vivo labeling of endogenous proteins with SMART delineates retina cellular and synaptic organization.

Nature communications, 16(1):3768.

A key application of CRISPR/Cas9-based genomic editing is modification of genes to introduce engineered sequences. However, the editing flexibility is severely constrained by the requirement for targeting sites in proximity to the desired modification site, which makes many modifications intractable. Here, we develop a strategy that overcomes this key limitation to allow CRISPR-based editing at any position with high efficiency. It relies on reconstructing the targeted gene using Silently Mutate And Repair Template (SMART) where we mutate the gap sequence in the repair template to prevent its base pairing with the target DNA while maintaining the same amino acid coding. Using vertebrate retina as a neuronal model system we document the application of SMART editing for labeling endogenous proteins in vivo with high efficiency. We show that SMART editing allows us to access numerous cell types in the retina and address fundamental cell biological questions pertaining to its organization. We propose that this approach will facilitate functional genomic studies in a wide range of systems and increase the precision of corrective gene therapies.

RevDate: 2025-04-22
CmpDate: 2025-04-23

Rengifo-Gonzalez M, Mazzuoli MV, Janssen AB, et al (2025)

Make-or-break prime editing for genome engineering in Streptococcus pneumoniae.

Nature communications, 16(1):3796.

CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.

RevDate: 2025-04-22

Silverstein RA, Kim N, Kroell AS, et al (2025)

Custom CRISPR-Cas9 PAM variants via scalable engineering and machine learning.

Nature pii:10.1038/s41586-025-09021-y [Epub ahead of print].

Engineering and characterizing proteins can be time-consuming and cumbersome, motivating the development of generalist CRISPR-Cas enzymes[1-4] to enable diverse genome editing applications. However, such enzymes have caveats such as an increased risk of off-target editing[3,5,6]. To enable scalable reprogramming of Cas9 enzymes, here we combined high-throughput protein engineering with machine learning (ML) to derive bespoke editors more uniquely suited to specific targets. Via structure/function-informed saturation mutagenesis and bacterial selections, we obtained nearly 1,000 engineered SpCas9 enzymes and characterized their protospacer-adjacent motif[7] (PAM) requirements to train a neural network that relates amino acid sequence to PAM specificity. By utilizing the resulting PAM ML algorithm (PAMmla) to predict the PAMs of 64 million SpCas9 enzymes, we identified efficacious and specific enzymes that outperform evolution-based and engineered SpCas9 enzymes as nucleases and base editors in human cells while reducing off-targets. An in silico directed evolution method enables user-directed Cas9 enzyme design, including for allele-selective targeting of the RHO P23H allele in human cells and mice. Together, PAMmla integrates ML and protein engineering to curate a catalog of SpCas9 enzymes with distinct PAM requirements, and motivates the use of efficient and safe bespoke Cas9 enzymes instead of generalist enzymes for various applications.

RevDate: 2025-04-23
CmpDate: 2025-04-22

Liu X, Wang P, Wang S, et al (2025)

The circular RNA circANK suppresses rice resistance to bacterial blight by inhibiting microRNA398b-mediated defense.

The Plant cell, 37(4):.

Circular RNAs (circRNAs) are prevalent in eukaryotic cells and have been linked to disease progressions. Their unique circular structure and stability make them potential biomarkers and therapeutic targets. Compared with animal models, plant circRNA research is still in its infancy. The lack of effective tools to specifically knock down circRNAs without affecting host gene expression has slowed the progress of plant circRNA research. Here, we have developed a CRISPR-Cas13d tool that can specifically knock down circRNAs in plant systems, successfully achieving the targeted knockdown of circRNAs in rice (Oryza sativa). We further focused on Os-circANK (a circRNA derived from Ankyrin repeat-containing protein), a circRNA differentially expressed in rice upon pathogen infection. Physiological and biochemical experiments revealed that Os-circANK functions as a sponge for miR398b, suppressing the cleavage of Cu/Zn-superoxidase dismutase (CSD)1/CSD2/copper chaperone for superoxide dismutase/superoxidase dismutaseX through competing endogenous RNA, leading to reduced reactive oxygen species levels following Xanthomonas oryzae pv. oryzae (Xoo) infection and a negative regulation of rice resistance to bacterial blight. Our findings indicate Os-circANK inhibits rice resistance to bacterial blight via the microRNA398b(miR398b)/CSD/SOD pathway.

RevDate: 2025-04-23
CmpDate: 2025-04-22

Cheng Y, Li G, Qi A, et al (2025)

A comprehensive all-in-one CRISPR toolbox for large-scale screens in plants.

The Plant cell, 37(4):.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) technologies facilitate routine genome engineering of one or a few genes at a time. However, large-scale CRISPR screens with guide RNA libraries remain challenging in plants. Here, we have developed a comprehensive all-in-one CRISPR toolbox for Cas9-based genome editing, cytosine base editing, adenine base editing (ABE), Cas12a-based genome editing and ABE, and CRISPR-Act3.0-based gene activation in both monocot and dicot plants. We evaluated all-in-one T-DNA expression vectors in rice (Oryza sativa, monocot) and tomato (Solanum lycopersicum, dicot) protoplasts, demonstrating their broad and reliable applicability. To showcase the applications of these vectors in CRISPR screens, we constructed guide RNA (gRNA) pools for testing in rice protoplasts, establishing a high-throughput approach to select high-activity gRNAs. Additionally, we demonstrated the efficacy of sgRNA library screening for targeted mutagenesis of ACETOLACTATE SYNTHASE in rice, recovering novel candidate alleles for herbicide resistance. Furthermore, we carried out a CRISPR activation screen in Arabidopsis thaliana, rapidly identifying potent gRNAs for FLOWERING LOCUS T activation that confer an early-flowering phenotype. This toolbox contains 61 versatile all-in-one vectors encompassing nearly all commonly used CRISPR technologies. It will facilitate large-scale genetic screens for loss-of-function or gain-of-function studies, presenting numerous promising applications in plants.

RevDate: 2025-04-22

Liu Y, Wu Y, Liu Y, et al (2025)

Arrest of CRISPR-Cas12a by Nonspecific Single-Stranded DNA for Biosensing.

Analytical chemistry [Epub ahead of print].

CRISPR-Cas technologies have emerged as powerful biosensing tools for the sensitive and specific detection of non-nucleic acid targets. However, existing biosensing strategies suffer from poor compatibility across diverse targets due to the complicated engineering of crRNA and DNA activator required for the CRISPR-Cas activity regulation. Herein, we report a novel and straightforward strategy for designing CRISPR-Cas12a-based biosensors that function by switching structures from single-stranded (ss)DNA/CRISPR-Cas12a assembly to DNA activator/CRISPR-Cas12a complex in the presence of target bacterium. The strategy begins with a ssDNA assembly made of a trans-acting RNA-cleaving DNAzyme (tRCD) and an RNA/DNA chimeric substrate (RCS). The ssDNA assembly has the ability to bind Cas12a nonspecifically, thus indeed blocking the CRISPR-Cas12a activity. By exploiting the specific recognition and cleavage capacities of tRCD for RCS in the presence of a target, the target-bound tRCD and the cleaved RCS are released from Cas12a, thus restoring the CRISPR-Cas12a activity. This method has been successfully applied for the sensitive (detection limit: 10[2] CFU/mL) detection of Escherichia coli (E. coli, EC) and Burkholderia gladioli (B. gladioli, BG). For the blind testing of 30 clinical urine samples, it exhibited 100% sensitivity and 100% specificity in identifying E. coli-associated urinary tract infections (UTIs).

RevDate: 2025-04-22

Liao X, Li Y, Wu Y, et al (2025)

Deep Learning-Based Classification of CRISPR Loci Using Repeat Sequences.

ACS synthetic biology [Epub ahead of print].

With the widespread application of the CRISPR-Cas system in gene editing and related fields, along with the increasing availability of metagenomic data, the demand for detecting and classifying CRISPR-Cas systems in metagenomic data sets has grown significantly. Traditional classification methods for CRISPR-Cas systems primarily rely on identifying cas genes near CRISPR arrays. However, in cases where cas gene information is absent, such as in metagenomes or fragmented genome assemblies, traditional methods may fail. Here, we present a deep learning-based method, CRISPRclassify-CNN-Att, which classifies CRISPR loci solely based on repeat sequences. CRISPRclassify-CNN-Att utilizes convolutional neural networks (CNNs) and self-attention mechanisms to extract features from repeat sequences. It employs a stacking strategy to address the imbalance of samples across different subtypes and uses transfer learning to improve classification accuracy for subtypes with fewer samples. CRISPRclassify-CNN-Att demonstrates outstanding performance in classifying multiple subtypes, particularly those with larger sample sizes. Although CRISPR loci classification traditionally depends on cas genes, CRISPRclassify-CNN-Att offers a novel approach that serves as a significant complement to cas-based methods, enabling the classification of orphan or distant CRISPR loci. The proposed tool is freely accessible via https://github.com/Xingyu-Liao/CRISPRclassify-CNN-Att.

RevDate: 2025-04-23
CmpDate: 2025-04-22

Wang T, Brown C, Doherty N, et al (2025)

Mannose and PMI depletion overcomes radiation resistance in HPV-negative head and neck cancer.

Cell communication and signaling : CCS, 23(1):189.

Radiotherapy is critical component of multidisciplinary cancer care, used as a primary and adjuvant treatment for patients with head and neck squamous cell carcinoma. This study investigates how mannose, a naturally occurring monosaccharide, combined with phosphomannose isomerase (PMI) depletion, enhances the sensitivity of HPV-negative head and neck tumour models to radiation. Isogenic PMI knockout models were generated by CRISPR/Cas9 gene editing, yielding a 20-fold increase in sensitivity to mannose in vitro, and causing significant tumour growth delay in vivo. This effect is driven by metabolic reprogramming, resulting in potent glycolytic suppression coupled with consistent depletion of ATP and glycolytic intermediates in PMI-depleted models. Functionally, these changes impede DNA damage repair following radiation, resulting in a significant increase in radiation sensitivity. Mannose and PMI ablation supressed both oxygen consumption rate and extracellular acidification, pushing cells towards a state of metabolic quiescence, effects contributing to increased radiation sensitivity under both normoxic and hypoxic conditions. In 3D-tumoursphere models, metabolic suppression by mannose and PMI depletion was shown to elevate intra-tumoursphere oxygen levels, contributing to significant in vitro oxygen-mediated radiosensitisation. These findings position PMI as a promising anti-tumour target, highlighting the potential of mannose as a metabolic radiosensitiser enhancing cancer treatment efficacy.

RevDate: 2025-04-23
CmpDate: 2025-04-21

Potlapalli BP, Dassau F, Fuchs J, et al (2025)

CRISPR-CISH: an in situ chromogenic DNA repeat detection system for research and life science education.

Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology, 33(1):7.

In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3' biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.

RevDate: 2025-04-21
CmpDate: 2025-04-21

Yang J, Song J, Feng Z, et al (2025)

Application of CRISPR-Cas9 in microbial cell factories.

Biotechnology letters, 47(3):46.

Metabolically engineered bacterial strains are rapidly emerging as a pivotal focus in the biosynthesis of an array of bio-based ingredients. Presently, CRISPR (clustered regularly interspaced short palindromic repeats) and its associated RNA-guided endonuclease (Cas9) are regarded as the most promising tool, having ushered in a transformative advancement in genome editing. Because of CRISPR-Cas9's accuracy and adaptability, metabolic engineers are now able to create novel regulatory systems, optimize pathways more effectively, and make extensive genome-scale alterations. Nevertheless, there are still obstacles to overcome in the application of CRISPR-Cas9 in novel microorganisms, particularly those industrial microorganism hosts that are resistant to traditional genetic manipulation techniques. How to further extend CRISPR-Cas9 to these microorganisms is an urgent problem to be solved. This article first introduces the mechanism and application of CRISPR-Cas9, and then discusses how to optimize CRISPR-Cas9 as a genome editing tool, including how to reduce off-target effects and how to improve targeting efficiency by optimizing design. Through offering a comprehensive perspective on the revolutionary effects of CRISPR-Cas9 in microbial cell factories, we hope to stimulate additional research and development in this exciting area.

RevDate: 2025-04-21
CmpDate: 2025-04-21

Filsinger GT, Mychack A, Lyerly E, et al (2025)

A diverse single-stranded DNA-annealing protein library enables efficient genome editing across bacterial phyla.

Proceedings of the National Academy of Sciences of the United States of America, 122(17):e2414342122.

Genome modification is essential for studying and engineering bacteria, yet making efficient modifications to most species remains challenging. Bacteriophage-encoded single-stranded DNA-annealing proteins (SSAPs) can facilitate efficient genome editing by homologous recombination, but their typically narrow host range limits broad application. Here, we demonstrate that a single library of 227 SSAPs enables efficient genome-editing across six diverse bacteria from three divergent classes: Actinomycetia (Mycobacterium smegmatis and Corynebacterium glutamicum), Alphaproteobacteria (Agrobacterium tumefaciens and Caulobacter crescentus), and Bacilli (Lactococcus lactis and Staphylococcus aureus). Surprisingly, the most effective SSAPs frequently originated from phyla distinct from their bacterial hosts, challenging the assumption that phylogenetic relatedness is necessary for recombination efficiency, and supporting the value of a large unbiased library. Across these hosts, the identified SSAPs enable genome modifications requiring efficient homologous recombination, demonstrated through three examples. First, we use SSAPs with Cas9 in C. crescentus to introduce single amino acid mutations with >70% efficiency. Second, we adapt SSAPs for dsDNA editing in C. glutamicum and S. aureus, enabling one-step gene knockouts using PCR products. Finally, we apply SSAPs for multiplexed editing in S. aureus to precisely map the interaction between a conserved protein and a small-molecule inhibitor. Overall, this library-based SSAP screen expands engineering capabilities across diverse, previously recalcitrant microbes, enabling efficient genetic manipulation for both fundamental research and biotechnological applications.

RevDate: 2025-04-23
CmpDate: 2025-04-23

Wang Q, Chen X, Li Y, et al (2025)

Protein-inorganic hybrid flowers with a two-stage accelerated strategy for stimulated activation of CRISPR/Cas12a enhance polynucleotide kinase biosensing.

Talanta, 292:127981.

Polynucleotide kinases (PNK) play a crucial role in DNA damage repair and are closely associated with specific diseases, making them promising targets for therapeutic intervention. In this study, we propose a two-stage accelerated strategy that utilizes protein-inorganic hybrid flowers (PHFs) to enhance the performance of the terminal deoxynucleotidyl transferase (TdT)-combined CRISPR/Cas12a system (TCS) for efficient detection of PNK activity. In TCS, the participation of PHFs confines the substrate probes (SPs) to a limited space, thereby significantly enhancing the local concentration of phosphorylated 3' termini of SPs and effectively promoting the enzymatic reaction kinetics as the first step in the accelerated strategy. Upon encountering the target PNK, the phosphorylated 3' termini were promptly recognized and dephosphorylated to 3'-OH termini. Subsequently, TdT catalyzed the assembly of deoxyadenosine triphosphates (dATPs) without a template, rapidly activating the CRISPR/Cas12a system by forming multiple polyadenine (poly-A) chains. PHF-fixed poly-A chains then substantially boosted the localized concentration of CRISPR/Cas12a systems and vastly enhanced their efficacy in cleaving reporter nucleic acids. Our findings indicated that the spatial confinement effect facilitated by PHFs promoted frequent molecular collisions and accelerated multiple enzymatic reactions. The developed sensing strategy allows for the detection of PNK activity within a linear range of 0.001-1 U/mL, with a detection limit of 1.82 × 10[-4] U/mL. Additionally, this strategy has been successfully applied to detect PNK activity in cell extracts and to screen for PNK inhibitors. Owing to these advantages, PNK can be rapidly and accurately detected with a high sensitivity, specificity, and biostability.

RevDate: 2025-04-23
CmpDate: 2025-04-23

Ling S, Zhang X, Dai Y, et al (2025)

Customizable virus-like particles deliver CRISPR-Cas9 ribonucleoprotein for effective ocular neovascular and Huntington's disease gene therapy.

Nature nanotechnology, 20(4):543-553.

In vivo CRISPR gene editing holds enormous potential for various diseases. Ideally, CRISPR delivery should be cell type-specific and time-restricted for optimal efficacy and safety, but customizable methods are lacking. Here we develop a cell-tropism programmable CRISPR-Cas9 ribonucleoprotein delivery system (RIDE) based on virus-like particles. The efficiency of RIDE was comparable to that of adeno-associated virus and lentiviral vectors and higher than lipid nanoparticles. RIDE could be readily reprogrammed to target dendritic cells, T cells and neurons, and significantly ameliorated the disease symptoms in both ocular neovascular and Huntington's disease models via cell-specific gene editing. In addition, RIDE could efficiently edit the huntingtin gene in patients' induced pluripotent stem cell-derived neurons and was tolerated in non-human primates. This study is expected to facilitate the development of in vivo CRISPR therapeutics.

RevDate: 2025-04-23
CmpDate: 2025-04-23

Huang X, Feng X, Yan YH, et al (2025)

Compartmentalized localization of perinuclear proteins within germ granules in C. elegans.

Developmental cell, 60(8):1251-1270.e3.

Germ granules, or nuage, are RNA-rich condensates that are often docked on the cytoplasmic surface of germline nuclei. C. elegans perinuclear germ granules are composed of multiple subcompartments, including P granules, Mutator foci, Z granules, SIMR foci, P -bodies, and E granules. Although many perinuclear proteins have been identified, their precise localization within the subcompartments of the germ granule is still unclear. Here, we systematically labeled perinuclear proteins with fluorescent tags via CRISPR-Cas9 technology. Using this nematode strain library, we identified a series of proteins localized in Z or E granules and extended the characterization of the D granule. Finally, we found that the LOTUS domain protein MIP-1/EGGD-1 regulated the multiphase organization of the germ granule. Overall, our work identified the germ-granule architecture and redefined the compartmental localization of perinuclear proteins. Additionally, the library of genetically modified nematode strains will facilitate research on C. elegans germ granules.

RevDate: 2025-04-22

Harshini P, Varghese R, Pachamuthu K, et al (2025)

Enhanced pigment production from plants and microbes: a genome editing approach.

3 Biotech, 15(5):129.

Pigments are known for their vital roles in the growth and development of plants and microbes. In addition, they are also an imperative component of several industries, including textiles, foods, and pharmaceuticals, owing to their immense colours and therapeutic potential. Conventionally, pigments are obtained from plant resources, and the advent of in-vitro propagation techniques boosted the massive production. However, it could not meet the booming demand, leading to the incorporation of new genetic engineering tools. This review focuses on the role of various genetic engineering techniques in enhancing pigment production in plants and microorganisms. It also critically analyzes the efficacy and bottlenecks of these techniques in augmenting pigment biosynthesis. Furthermore, the use of microbes as pigment biofactories and the prospects in the field of genome editing to augment pigment synthesis are discussed. The limitations in the existing techniques underline the need for advanced genome editing strategies to broaden the mass production of pigments to meet the surging needs.

RevDate: 2025-04-22
CmpDate: 2025-04-19

Hurtado JE, Schieferecke AJ, Halperin SO, et al (2025)

Nickase fidelity drives EvolvR-mediated diversification in mammalian cells.

Nature communications, 16(1):3723.

In vivo genetic diversifiers have previously enabled efficient searches of genetic variant fitness landscapes for continuous directed evolution. However, existing genomic diversification modalities for mammalian genomic loci exclusively rely on deaminases to generate transition mutations within target loci, forfeiting access to most missense mutations. Here, we engineer CRISPR-guided error-prone DNA polymerases (EvolvR) to diversify all four nucleotides within genomic loci in mammalian cells. We demonstrate that EvolvR generates both transition and transversion mutations throughout a mutation window of at least 40 bp and implement EvolvR to evolve previously unreported drug-resistant MAP2K1 variants via substitutions not achievable with deaminases. Moreover, we discover that the nickase's mismatch tolerance limits EvolvR's mutation window and substitution biases in a gRNA-specific fashion. To compensate for gRNA-to-gRNA variability in mutagenesis, we maximize the number of gRNA target sequences by incorporating a PAM-flexible nickase into EvolvR. Finally, we find a strong correlation between predicted free energy changes underlying R-loop formation and EvolvR's performance using a given gRNA. The EvolvR system diversifies all four nucleotides to enable the evolution of mammalian cells, while nuclease and gRNA-specific properties underlying nickase fidelity can be engineered to further enhance EvolvR's mutation rates.

RevDate: 2025-04-19
CmpDate: 2025-04-19

Xin Y, Guo T, M Qiao (2025)

Current application and future prospects of CRISPR-Cas in lactic acid Bacteria: A review.

Food research international (Ottawa, Ont.), 209:116315.

Lactic acid bacteria (LABs) have a long history of use in food and beverages fermentation. Recently, several LABs have gained attention as starter or non-starter cultures and probiotics for making functional fermented foods, which have the potential to enhance human health. In addition, certain LABs show great potential as microbial cell factories for producing food-related chemicals. However, enhancing the outcomes of starter and non-starter cultures, exploring the complicated probiotic mechanism of LABs, and engineering strains to enhance the yields of high-value compounds for precision fermentation remains challenging due to the time-consuming and labor-intensive current genome editing tools. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) system, originally an adaptive immune system in bacteria, has revolutionized genome editing, metabolic engineering and synthetic biology. Its versatility has resulted in extensive applications across diverse organisms. The widespread distribution of CRISPR-Cas systems and the diversity of CRISPR arrays in LAB genomes highlight their potential for studying the evolution of LABs. This review discusses the current advancement of CRISPR-Cas systems in engineering LABs for food application. Moreover, it outlines future research directions aimed at harnessing CRISPR-Cas systems to advance lactic acid bacterial research and drive innovation in food science.

RevDate: 2025-04-19

Zou W, Huo B, Tu Y, et al (2025)

Metabolic reprogramming by chemo-gene co-delivery nanoparticles for chemo-immunotherapy in head and neck squamous cell carcinoma.

Acta biomaterialia pii:S1742-7061(25)00272-7 [Epub ahead of print].

The therapeutic effects of platinum-based drugs are closely linked to the dysregulation of tumor metabolic-immune microenvironment, particularly aberrant lactate accumulation. Herein, we engineered multifunctional nanoparticles (PPPt[IV] NPs) through electrostatic self-assembly of poly(β-amino ester) to co-encapsulate the cisplatin prodrug (Pt[IV]) and CRISPR/Cas9-PKM2 plasmids. Mechanistically, PPPt[IV] NPs efficiently entered cells via endocytosis, followed by escape from lysosomal degradation and cargo release. The reduction of Pt[IV] prodrug to the active Pt[II] via GSH depletion induced DNA damage and ROS upregulation, thereby triggering apoptosis. Concurrently, CRISPR/Cas9-mediated PKM2 knockdown suppressed the Warburg effect, resulting in reduced lactate production and downregulated expression of HIF-1α and PD-L1. These alterations drove immune microenvironment remodeling through enhanced dendritic cell maturation, polarized M1 macrophages, and altered cytokine profiles (characterized by upregulation of IFN-γ, TNF-α, and IL-12 alongside suppression of IL-10), ultimately activating T cell-mediated antitumor immunity. Compared to conventional cisplatin, PPPt[IV] NPs demonstrated superior efficacy against both primary and recurrent tumors while reducing nephrotoxicity through synergistic chemo-immunotherapeutic effects, offering a valuable strategy for HNSCC treatment. STATEMENT OF SIGNIFICANCE: This study engineered an innovative nanoplatform (PPPt[IV]) that synergistically integrates a Pt[IV] prodrug with a CRISPR/Cas-PKM2 plasmid for treating head and neck squamous cell carcinoma. By simultaneously enhancing DNA damage and reversing lactate-mediated immunosuppression, PPPt[IV] nanoplatform achieved chemo-immunotherapy that showed greater suppression of primary and recurrent tumors with reduced renal toxicity compared to cisplatin. This nanotechnology-driven strategy provides valuable insights for the combination of platinum-based drugs with immunometabolic interventions.

RevDate: 2025-04-21
CmpDate: 2025-04-18

Gonzalez E, Anderson MAE, Ang JXD, et al (2025)

Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi.

Scientific reports, 15(1):13408.

Anopheles stephensi, a major Asian malaria vector, is invading Africa and has been implicated in recent outbreaks of urban malaria. Control of this species is key to eliminating malaria in Africa. Genetic control strategies, and CRISPR/Cas9-based gene drives are emerging as promising species-specific, environmentally friendly, scalable, affordable methods for pest control. To implement these strategies, a key parameter to optimize for high efficiency is the spatiotemporal control of Cas9 and the gRNA. Here, we assessed the ability of four RNA Pol III promoters to bias the inheritance of a gene drive element inserted into the cd gene of An. stephensi. We determined the homing efficiency and examined eye phenotype as a proxy for non-homologous end joining (NHEJ) events in somatic tissue. We found all four promoters to be active, with mean inheritance rates up to 99.8%. We found a strong effect of the Cas9-bearing grandparent (grandparent genotype), likely due to maternally deposited Cas9.

RevDate: 2025-04-18

Huang W, Ruyechan MC, KS Ralston (2025)

Work with me here: variations in genome content and emerging genetic tools in Entamoeba histolytica.

Trends in parasitology pii:S1471-4922(25)00074-1 [Epub ahead of print].

Entamoeba histolytica is the causative agent of amoebiasis, a significant source of morbidity and mortality in developing nations. Despite this, E. histolytica is understudied, leading to few treatment options and a poor understanding of pathogenesis. Genetic tools have historically been limited. By applying modern approaches, it was recently revealed that the genome is aneuploid. Interestingly, gene expression levels do not correlate with ploidy, potentially highlighting the importance of RNAi in gene regulation. Characterization of the RNAi pathway has led to potent tools for targeted gene knockdown, and the advent of RNAi-based forward genetics. CRISPR/Cas tools for editing the endogenous genome are an exciting possibility on the horizon. We celebrate the gains that have made E. histolytica tractable and anticipate continued advances.

RevDate: 2025-04-18
CmpDate: 2025-04-18

Eidelman M, Eisenberg E, EY Levanon (2025)

Global quantification of off-target activity by base editors.

Methods in enzymology, 713:255-270.

Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.

RevDate: 2025-04-22
CmpDate: 2025-04-22

Wu Q, Li S, Long X, et al (2025)

β-Galactosidase-Mediated, Mn[2+]-Activated CRISPR/Cas12a Cascade Reaction for Immunosorbent Assay of Carbendazim.

Analytical chemistry, 97(15):8402-8410.

The CRISPR/Cas12a system is an emerging enzymatic tool for the development of enzyme-linked immunosorbent assay (ELISA) methods, owing to its robust signal amplification capability. Currently, most CRISPR/Cas12a-based ELISA approaches rely on strategies that convert target detection into nucleic acid analysis. This report presents a novel enzymatic cascade reaction for signal transduction and amplification in the development of a CRISPR/Cas12a-based ELISA method, utilizing β-galactosidase (β-gal)-mediated activation of the CRISPR/Cas12a system. Carbendazim (CBD), a widely used and versatile broad-spectrum benzimidazole fungicide, was chosen as the model analyte. In the absence of CBD, streptavidin-labeled β-gal is captured by a biotinylated secondary antibody immobilized on the microplate. The captured β-gal catalyzes the hydrolysis of p-aminophenyl β-D-galactopyranoside to generate p-aminophenol. This compound subsequently facilitates the decomposition of MnO2 nanosheets, leading to the generation of Mn[2+] ions. The Mn[2+] ions modulate the activity of the CRISPR/Cas12a system, thus producing high fluorescence in the detection solution. In the presence of CBD, the amount of β-gal captured on the microplate is reduced, thereby preventing effective cleavage of the reporter molecule by Cas12a, which results in a low fluorescence signal. After systematically optimizing experimental conditions, the developed method successfully detected CBD, demonstrating high sensitivity, selectivity, and applicability in complex food matrices. In comparison to the traditional nucleic acid-activated CRISPR/Cas12a-based ELISA method, our approach, which integrates β-gal-mediated, Mn[2+]-activated CRISPR/Cas12a cascade reactions into ELISA, exhibits superior analytical performance, thereby broadening the applicability of CRISPR/Cas12a for sensitive and convenient small-molecule analysis.

RevDate: 2025-04-22
CmpDate: 2025-04-22

Cheng HJ, Liu Y, Li HD, et al (2025)

Catalytic hairpin assembly-coupled CRISPR/Cas12a biosensor for sensitive detection of melamine in dairy products.

Chemical communications (Cambridge, England), 61(34):6300-6303.

We combined catalytic hairpin assembly (CHA) with the Cas12a system for detecting melamine adulteration. This system involved two-step signal conversion and two-level amplification, boosting the sensor's versatility and sensitivity. The sensor showed excellent specificity and applicability for melamine detection in dairy products, and was broadened to viral nucleic acid detection.

RevDate: 2025-04-22
CmpDate: 2025-04-22

Yang S, Ren L, Fan N, et al (2025)

CRISPR-Cas12a with split crRNA for the direct and sensitive detection of microRNA.

The Analyst, 150(9):1884-1890.

microRNAs (miRNAs) have been identified as potential biomarkers. Despite the prevalence of quantitative PCR in the field of miRNA detection, this technology is encumbered by the complexity of its methodology. This study presents a novel CRISPR/Cas12a-based method for the direct and sensitive detection of miRNA-21 using split crRNA. The system comprises Cas12a protein, crRNA-handle, and activator DNA complementary to the target miRNA. In the presence of the target miRNA, it binds to the activator DNA, forming a duplex. The formed duplex, in conjunction with the crRNA-handle, activates Cas12a's trans-cleavage activity. This leads to cleavage of a fluorescent reporter, generating an enhanced signal. The method enables direct RNA detection without reverse transcription or sample amplification, offering simplicity and efficiency. This method demonstrates high sensitivity with a minimum detectable limit of 5 pM. Furthermore, the method's specificity is substantiated by its capacity to discern point mutations in miRNA. This system has been shown to quantitatively analyse miRNA-21 levels present within serum, as evidenced by the recovery experiment. Therefore, the method's simplicity, stability, and cost-effectiveness render it a powerful tool for nucleic acid detection, with potential for clinical applications.

RevDate: 2025-04-22
CmpDate: 2025-04-22

He S, Lin W, Liu X, et al (2025)

A DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor for sensitive detection of Pb[2+] based on DNAzymes.

The Analyst, 150(9):1778-1784.

Lead pollution presents a significant threat to ecological systems and human health, underscoring the urgent need for highly sensitive detection methods. Herein, we introduce a novel DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor (MDD-Cas12a) for sensitive detection of Pb[2+] based on DNAzymes. To accomplish this, we designed a substrate strand containing a long DNA concatemer encoding multiple protospacer adjacent motifs (PAMs) and protospacer sequences for activation of the CRISPR/Cas12a system. The DNA concatemer was subsequently anchored to the surface of magnetic beads (MBs) to fabricate a MBs-DNA concatemer nanoprobe. In the presence of Pb[2+], the DNAzyme structure catalyzes the cleavage of the substrate strand, leading to the release of DNA concatemers. Following magnetic separation, the released DNA concatemers significantly activate the non-specific trans-cleavage activity of the Cas12a/crRNA complex. The fluorescence reporter DNA is then completely cleaved by the activated Cas12a/crRNA complex, and the Pb[2+] concentration in the sample can be quantified by measuring the fluorescence signal. By harnessing the specific recognition capability of DNAzymes for Pb[2+], the programmability of DNA concatemers, and the self-amplification features of the CRISPR/Cas12a system, the MDD-Cas12a platform demonstrates high sensitivity and specificity for detecting Pb[2+] in milk and lake water samples.

RevDate: 2025-04-22
CmpDate: 2025-04-22

Wiley L, Cheek M, LaFar E, et al (2025)

The Ethics of Human Embryo Editing via CRISPR-Cas9 Technology: A Systematic Review of Ethical Arguments, Reasons, and Concerns.

HEC forum : an interdisciplinary journal on hospitals' ethical and legal issues, 37(2):267-303.

The possibility of editing the genomes of human embryos has generated significant discussion and interest as a matter of science and ethics. While it holds significant promise to prevent or treat disease, research on and potential clinical applications of human embryo editing also raise ethical, regulatory, and safety concerns. This systematic review included 223 publications to identify the ethical arguments, reasons, and concerns that have been offered for and against the editing of human embryos using CRISPR-Cas9 technology. We identified six major themes: risk/harm; potential benefit; oversight; informed consent; justice, equity, and other social considerations; and eugenics. We explore these themes and provide an overview and analysis of the critical points in the current literature.

RevDate: 2025-04-22
CmpDate: 2025-01-22

Pandey S, Gao XD, Krasnow NA, et al (2025)

Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.

Nature biomedical engineering, 9(1):22-39.

Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites, PASSIGE with eeBxb1 led to an average targeted-gene-integration efficiencies of 23% (4.2-fold that of wild-type Bxb1). Notably, integration efficiencies exceeded 30% at multiple sites in primary human fibroblasts. PASSIGE with evoBxb1 or eeBxb1 outperformed PASTE (for 'programmable addition via site-specific targeting elements', a method that uses prime editors fused to recombinases) on average by 9.1-fold and 16-fold, respectively. PASSIGE with continuously evolved recombinases is an unusually efficient method for the targeted integration of genes in mammalian cells.

RevDate: 2025-04-18

Gaba S, Sahu M, Chauhan N, et al (2025)

Transforming growth factor alpha: Key insights into physiological role, cancer therapeutics, and biomarker potential (A review).

International journal of biological macromolecules pii:S0141-8130(25)03764-X [Epub ahead of print].

Transforming Growth Factor Alpha (TGF-α) is a critical member of the epidermal growth factor (EGF) family and a key regulator of various physiological processes, including cellular proliferation, survival, differentiation, wound repair, and tissue regeneration. Deficiencies or mutations in TGF-α have been associated with impaired tissue development and organ growth, underscoring its critical role in maintaining normal and healthy physiology. Alterations in its levels are frequently implicated in the neoplastic transformation of cells, contributing to cancer development. Several strategies for targeting TGF-α in cancer therapy have been explored, such as the use of antibodies, recombinant proteins, oligonucleotide-mediated interference in ligand synthesis, ligand sequestration via binding proteins, and modulation of the signal transduction pathway. Furthermore, there is growing interest in the potential of TGF-α as a diagnostic or prognostic biomarker for cancer. This review delves into the role of TGF-α in normal physiology and its involvement in carcinogenesis. It highlights therapies targeting TGF-α and explores future directions in targeting TGF-α /EGFR signaling using advancing approaches, including nanoparticle-based drug delivery systems, CRISPR-Cas genome editing tool, PROTAC, and combination therapies. By bringing attention to this molecule, we aim to explore its untapped potential in cancer treatment and inspire further research into its promising applications across related fields. While recent studies highlight the promise of TGF-α as a clinical biomarker, further research is needed to validate its specificity and integration into personalized medicine. By providing a comprehensive overview of TGF-α in both normal and pathological contexts, this review aims to offer new insights into its translational applications in cancer therapeutics and biomarker discovery.

RevDate: 2025-04-21
CmpDate: 2025-04-18

Wang F, Chen Y, Huang R, et al (2025)

Identification of SURF4 and RALGAPA1 as promising therapeutic targets in glioblastoma and pan-cancer through integrative multi-omics, CRISPR-Cas9 screening and prognostic meta-analysis.

Cancer immunology, immunotherapy : CII, 74(6):175.

Glioblastoma (GBM) is the most aggressive and malignant type of primary brain tumor, with a median survival time of less than two years and a uniformly poor prognosis, despite multimodal therapeutic approaches, which highlights an urgent need for novel therapeutic targets. In this study, by integrative multi-omics analysis from CPTAC database, DepMap database and seven independent GBM cohorts, four hub genes (CD44, SURF4, IGSF3 and RALGAPA1) were identified as essential genes regulated by cancer driver genes with robust prognostic value. GBM multi-omics data from public and in-house cohorts validated that CD44 and SURF4 might be synthetic lethal partners of loss-of-function tumor suppressor genes. Analysis for immune-related pathway activity revealed complex regulation relationships of the four hub genes in tumor microenvironment (TME). Further investigation on SURF4 in pathway activity, immune therapy response and drug sensitivity proposed that SURF4 emerged as a promising therapeutic target for GBM, even for pan-cancer. Pan-cancer multi-omics exploration suggested that RALGAPA1 may be a tumor suppressor gene. By screening the first-generation and second-generation DepMap database, four genes (CCDC106, GAL3ST1, GDI2 and HSF1) might be considered as synthetic targets after mutation of RALGAPA1 as a tumor suppressor gene.

RevDate: 2025-04-20
CmpDate: 2025-04-17

Loughran AJ, Narina S, Klein J, et al (2025)

Rapid and robust validation of pooled CRISPR knockout screens using CelFi.

Scientific reports, 15(1):13358.

Pooled CRISPR screens are vital in the unbiased interrogation of gene function and are instrumental in uncovering therapeutic targets and biological processes. However, follow-up hit validation is critical to confirm observed results. Researchers need a simple and robust approach to rapidly verify putative hits and test resulting observations. Thus, we developed a CRISPR-based method for hit validation that tests the effect of a genetic perturbation on cell fitness. By editing target loci and monitoring the indel profiles over time, we have created a Cellular Fitness (CelFi) assay that can elucidate cellular vulnerabilities and verify hits from pooled CRISPR knockout screens. Unlike traditional cellular fitness assays that evaluate viability over time, the CelFi assay correlates changes in the indel profile at the target gene with a selective growth advantage or disadvantage in individual cells over time. Moreover, the CelFi assay can be utilized to evaluate gene dependencies and test new hypotheses, regardless of variations in single guide RNA optimization, ribonucleoprotein concentration, and gene copy number.

RevDate: 2025-04-20
CmpDate: 2025-04-17

Nakamura K, Aoyama-Ishiwatari S, Nagao T, et al (2025)

Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering.

Nature communications, 16(1):3401.

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identify the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-electron tomography, and correlative light-electron microscopy. Single molecule tracking reveals highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and captures at MERCS. Overexpression of FKBP8 is sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrate their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

RevDate: 2025-04-17
CmpDate: 2025-04-17

Wang X, Yue Y, Zhai Y, et al (2025)

Functional redundancy in the toxic pathway of Bt protein Cry1Ab, but not Cry1Fa, against the Asian corn borer.

Proceedings of the National Academy of Sciences of the United States of America, 122(16):e2503674122.

Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been used extensively to control some major crop pests, but their benefits decrease when pests evolve resistance. Better understanding of the genetic basis of resistance is needed to effectively monitor, manage, and counter pest resistance to Bt crops. Resistance to Bt proteins in at least 11 species of Lepidoptera, including many important crop pests, is associated with naturally occurring mutations that disrupt one or more of three larval midgut proteins: cadherin and ATP-binding cassette proteins ABCC2 and ABCC3. Here, we determined how CRISPR/Cas9-mediated mutations disrupting cadherin, ABCC2, and ABCC3 singly and in pairs affect resistance to Bt proteins Cry1Ab and Cry1Fa in the Asian corn borer (Ostrinia furnacalis), which is the most damaging pest of corn in Asia and is closely related to the European corn borer (Ostrinia nubilalis), a major pest in Europe and North America. The results from bioassays of six knockout strains and their parent susceptible strain support a model in which Cry1Ab can kill larvae via one path requiring ABCC2 or another path requiring cadherin and ABCC3, whereas Cry1Fa uses only the first path. The model's predictions are generally supported by results from genetic linkage analyses and responses to Cry1Ab and Cry1Fa of Sf9 cells and Xenopus oocytes modified to produce cadherin, ABCC2, and ABCC3 singly or in pairs. The functional redundancy identified here for Cry1Ab could sustain its efficacy against O. furnacalis and may exemplify a widespread natural strategy for delaying resistance.

RevDate: 2025-04-21
CmpDate: 2025-04-21

Miskel D, Kurzella J, Rings F, et al (2025)

Functional COPA is indispensable for early embryo development beyond major genome activation in bovines.

Theriogenology, 241:117415.

Embryonic genome activation is divided into a minor and a major wave of transition to endogenous transcription. In bovines, minor genome activation begins early in the 2-cell stage and is completed by the 8-cell stage when major genome activation becomes dominant. While the activation of genes known to regulate early development have been studied extensively, genes involved in more central cellular functions have not been examined. Taking advantage of the CRISPR Cas9 system, the present study investigated the effect of knocking out the Golgi retrograde protein transporter COPA on early bovine development. After the electroporation of presumptive zygotes with Cas9 ribonucleoproteins targeting COPA exon 6, sequences of 2 (11 %) and 4-cell (16 %) embryos showed knockouts of COPA whereas 8-cell embryos and blastocysts did not, demonstrating that COPA is necessary for development to the 8-cell stage and beyond. Using a repair template containing silent mutations along the target site, COPA loss of wildtype was observed in 5 blastocysts, with successful knock-in of the template on at least one allele. This shows that an edited yet functional copy of COPA can save the developmental capacity of the embryo and demonstrates that Cas9 activity at the target region itself is not responsible for the loss of function. Together, the present study revealed that COPA is necessary for embryonic development, and that the timing of this necessity is before major genome activation onset. More generally, this study further demonstrates the utility of genome editing within reproductive biotechnology for the interrogation of gene function and early embryonic development.

RevDate: 2025-04-21
CmpDate: 2025-04-21

Zhang X, Van Treeck B, Horton CA, et al (2025)

Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.

Nature biotechnology, 43(1):42-51.

Current approaches for inserting autonomous transgenes into the genome, such as CRISPR-Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.

RevDate: 2025-04-19
CmpDate: 2025-04-17

Bono H (2025)

Recent Advances in Genome Editing and Bioinformatics: Addressing Challenges in Genome Editing Implementation and Genome Sequencing.

International journal of molecular sciences, 26(7):.

Genome-editing technology has advanced significantly since the 2020 Nobel Prize in Chemistry was awarded for the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). While CRISPR-Cas9 has become widely used in academic research, its social implementation has lagged due to unresolved patent disputes and slower progress in gene function analysis. To address this, new approaches bypassing direct gene function analysis are needed, with bioinformatics and next-generation sequencing (NGS) playing crucial roles. NGS is essential for sequencing the genome of target species, but challenges such as data quality, genome heterogeneity, ploidy, and small individual sizes persist. Despite these issues, advancements in sequencing technologies, like PacBio high-fidelity (HiFi) long reads and high-throughput chromosome conformation capture (Hi-C), have improved genome sequencing. Bioinformatics contributes to genome editing through off-target prediction and target gene selection, both of which require accurate genome sequence information. In this review, I will give updates on the development of genome editing and bioinformatics technologies with a focus on the rapid progress in genome sequencing.

RevDate: 2025-04-19
CmpDate: 2025-04-17

Fan S, Jia L, Wu J, et al (2025)

Harnessing the Potential of CRISPR/Cas in Targeted Alfalfa Improvement for Stress Resilience.

International journal of molecular sciences, 26(7):.

Alfalfa (Medicago sativa), recognized as the most valuable legume feed crop, faces significant challenges in enhancing both qualitative and quantitative production amidst the pressures of climate change. This review highlights these challenges, including the underutilization of genomic and genetic resources, while proposing potential solutions through genome editing. Our focus is on leveraging CRISPR/Cas technology in conjunction with decades of advancements in conventional breeding to expedite the improvement of alfalfa. By adopting this approach, we aim to overcome the limitations of traditional alfalfa improvement approaches and accelerate the development of improved cultivars capable of thriving in changing climates. Key candidate traits for CRISPR/Cas genome editing, as reviewed in the latest literature, include nutrient use efficiency, freezing tolerance, and resistance to pests and diseases. We dissect literature on potential gene pathways associated with these traits, providing molecular breeders with valuable insights for utilizing CRISPR/Cas genome editing. Furthermore, we propose editing modalities to expedite the development of stress-resilient, genome-edited alfalfa that can effectively cope with climate change.

RevDate: 2025-04-19
CmpDate: 2025-04-17

Zanatta CB, Narendja F, El Jawhary H, et al (2025)

Suitability of Real-Time PCR Methods for New Genomic Technique Detection in the Context of the European Regulations: A Case Study in Arabidopsis.

International journal of molecular sciences, 26(7):.

PCR methods are widely applied for the detection of genetically modified organisms (GMOs) in Europe, facilitating compliance with stringent regulatory requirements and enabling the accurate identification and quantification of genetically modified traits in various crops and foodstuffs. This manuscript investigates the suitability of real-time PCR methods for detecting organisms generated through new genomic techniques (NGTs), specifically focusing on a case study using Arabidopsis thaliana as a model gene-edited plant. Given the complexities of European regulations regarding genetically modified organisms (GMOs) and the classification of gene-edited plants, there is a pressing need for robust detection methods. Our study highlights the development and validation of a novel single-plex real-time PCR method targeting a specific single nucleotide polymorphism (SNP) in the grf1-3 gene modified using CRISPR-Cas9 technology. We emphasize the effectiveness of locked nucleic acid (LNA)-modified primers in improving specificity. The results demonstrate that while the grf1-3 LNA method successfully detected and quantified gene-edited Arabidopsis DNA, achieving absolute specificity remains a challenge. This study also addresses the significance of the cross-laboratory method for validation, demonstrating that the method developed for an SNP-modified allele can be performed in accordance with the precision and trueness criteria established by the European Network of GMO Laboratories (ENGL). Furthermore, we call for continued collaboration among regulatory agencies, academia, and industry stakeholders to refine detection strategies. This proactive approach is essential not only for regulatory compliance but also for maintaining public trust in the safe integration of gene-edited organisms into food products.

RevDate: 2025-04-19
CmpDate: 2025-04-19

Ito R, Nakano T, Sugawara A, et al (2025)

A CRISPR-based high-throughput screening system identifies bromodomain inhibitors as transcriptional suppressors of CYP11B1.

Biochemical and biophysical research communications, 762:151779.

CYP11B1 encodes steroid 11β-hydroxylase, the final rate-limiting enzyme for cortisol biosynthesis in the adrenal cortex. Excessive cortisol production is a hallmark of Cushing's disease (CD). While direct enzymatic inhibitors have been explored, achieving specificity remains a challenge due to the high homology between CYP11B1 and CYP11B2, highlighting transcriptional suppression of CYP11B1 as an alternative therapeutic strategy. To identify transcriptional regulators of CYP11B1, we generated genome-edited H295R adrenal cells carrying a luciferase reporter knocked into the endogenous CYP11B1 locus. Using this reporter cell line, we established a high-throughput screening (HTS) platform and screened a focused chemical library targeting epigenetic-related factors, given the importance of epigenetic mechanisms in gene regulation. Among eight candidate compounds identified, we focused on JQ1, a bromodomain inhibitor. JQ1 significantly suppressed Forskolin-induced CYP11B1 promoter activity and mRNA expression without causing cytotoxicity, suggesting the involvement of epigenetic readers in the transcriptional regulation of steroidogenic genes. Furthermore, the reporter-based HTS platform developed here, when combined with our previously established CYP11B2-luciferase system, may facilitate the identification of compounds that selectively modulate adrenal steroidogenic pathways. These findings provide a foundation for the development of novel transcription-targeted therapies for CD.

RevDate: 2025-04-08

Tang S, Žedaveinytė R, Burman N, et al (2025)

Protein-primed DNA homopolymer synthesis by an antiviral reverse transcriptase.

bioRxiv : the preprint server for biology.

Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign DNA for degradation. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by leveraging DNA synthesis instead. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene, leading to expression of an antiviral effector protein, Neo. It remains unknown whether similar mechanisms of defense are employed by other DRT families. Focusing on DRT9, here we uncover an unprecedented mechanism of DNA homopolymer synthesis, in which viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell to drive abortive infection and population-level immunity. Cryo-EM structures reveal how a conserved noncoding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and RNA-templated poly-dA synthesis. Remarkably, biochemical and functional experiments identify conserved tyrosine residues within the reverse transcriptase itself that prime DNA synthesis, leading to the formation of high-molecular weight protein-DNA covalent adducts. Synthesis of poly-dA in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers of DRT9. Collectively, our work unveils a novel nucleic acid-driven defense system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.

RevDate: 2025-04-19
CmpDate: 2025-04-19

Li X, Wang Z, Man X, et al (2025)

Research advances CRISPR gene editing technology generated models in the study of epithelial ovarian carcinoma.

Gynecologic oncology, 195:34-44.

Epithelial ovarian carcinoma (EOC), the most lethal gynecologic cancer, is often diagnosed at advanced stages, which urge us to explore the novel therapeutic strategies. Mouse models have played a crucial role in elucidating the molecular mechanisms for the development ovarian cancer and its therapeutic strategies. However, there are still various challenges in modeling the genetic drivers of ovarian cancer in animal models. Here, we provided an overview of the research advances for the molecular mechanisms underlying EOC development, therapeutic strategies, the CRISPR genome editing technology and its generated EOC models. The review also comprehensively discussed the advantages and obstacles of CRISPR in generating EOC mouse models and the promising therapeutic approach by correcting the oncogenes of EOC through in vivo delivery of gene-edited components. The development of more precise animal models, along with a deeper understanding of EOC molecular mechanisms, will dramatically benefit the investigation and treatment of EOC.

RevDate: 2025-04-17
CmpDate: 2025-04-17

Ciapaite J, Albersen M, Savelberg SMC, et al (2025)

Broad Vitamin B6-Related Metabolic Disturbances in a Zebrafish Model of Hypophosphatasia (TNSALP-Deficiency).

International journal of molecular sciences, 26(7):.

Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B6-responsive seizures. To study HPP, we generated alpl[-/-] zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl[-/-] compared to alpl[+/+] embryos. The survival of alpl[-/-] zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl[-/-] embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl[-/-] embryos. Accumulation of d3-pyridoxal 5'-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl[-/-] embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl[-/-] embryos. Metabolic profiling of alpl[-/-] larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B6-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments.

RevDate: 2025-04-17
CmpDate: 2025-04-17

He J, Liu J, Yue Y, et al (2025)

Genome Editing in Mouse Embryo Using the CRISPR/Cas12i3 System.

International journal of molecular sciences, 26(7):.

The CRISPR/Cas system is a sizable family that is currently a popular and efficient gene editing tool. Cas12i3, as a member of the Type V-I family, has the characteristics of recognizing T-rich PAM sequences and being guided by shorter crRNA and has higher gene editing efficiency than Cas9 in rice. However, as a potential tool in accelerating the breeding process, the application of Cas12i3 in mammalian embryos has not yet been reported. Our study systematically evaluated the feasibility of applying CRISPR/Cas12i3 to gene editing in mouse embryos, with the core pluripotency regulator gene Nanog as the target. We successfully constructed a Nanog loss-of-function mouse embryo model using CRISPR/Cas12i3. At the targeted Nanog locus, its editing efficiency exceeded that of the Cas9 system under matched experimental conditions; no off-target phenomenon was detected. Moreover, the Cas12i3 system exhibited no side effect on mouse embryo development and proliferation of blastocyst cells. Finally, we obtained healthy chimeric gene-edited offspring by optimizing the concentration of the Cas12i3 mixture. These results confirm the feasibility and safety of CRISPR/Cas12i3 for gene editing in mammals, which provides a reliable tool for one-step generation of gene-edited animals for applications in biology, medical research, and large livestock breeding.

RevDate: 2025-04-17
CmpDate: 2025-04-17

Daliri K, Hescheler J, Newby GA, et al (2025)

Modulating Collagen I Expression in Fibroblasts by CRISPR-Cas9 Base Editing of the Collagen 1A1 Promoter.

International journal of molecular sciences, 26(7):.

Fibrotic diseases, contributing to a significant portion of global mortality, highlight the need for innovative therapies. This study explores a novel approach to disrupt the expression of collagen by using adenine base editing to target Col1a1, a key gene driving both fibrosis and cancer metastasis. Editing Col1a1 in fibroblasts demonstrated 18% editing efficiency. An analysis of a specific clone harboring a CCAAT-to-CCGGA mutation in the Col1a1 promoter revealed reduced collagen production. Notably, when wild-type fibroblasts were cultured on the Col1a1-edited matrix, no compensatory collagen upregulation was detected, suggesting a lack of feedback mechanism in fibroblasts. Furthermore, the matrix derived from edited fibroblasts did not support the growth of MCF-7 cancer cells. These findings suggest that Col1a1 gene editing holds promise as a potential therapeutic strategy for fibrotic diseases. Further investigation is warranted to fully elucidate the implications of these findings for fibrosis and cancer.

RevDate: 2025-04-18
CmpDate: 2025-04-17

Kim T, Park BS, Heo S, et al (2025)

Combinatorial CRISPR screen reveals FYN and KDM4 as targets for synergistic drug combination for treating triple negative breast cancer.

eLife, 13:.

Tyrosine kinases play a crucial role in cell proliferation and survival and are extensively investigated as targets for cancer treatment. However, the efficacy of most tyrosine kinase inhibitors (TKIs) in cancer therapy is limited due to resistance. In this study, we identify a synergistic combination therapy involving TKIs for the treatment of triple negative breast cancer. By employing pairwise tyrosine kinase knockout CRISPR screens, we identify FYN and KDM4 as critical targets whose inhibition enhances the effectiveness of TKIs, such as NVP-ADW742 (IGF-1R inhibitor), gefitinib (EGFR inhibitor), and imatinib (ABL inhibitor) both in vitro and in vivo. Mechanistically, treatment with TKIs upregulates the transcription of KDM4, which in turn demethylates H3K9me3 at FYN enhancer for FYN transcription. This compensatory activation of FYN and KDM4 contributes to the resistance against TKIs. FYN expression is associated with therapy resistance and persistence by demonstrating its upregulation in various experimental models of drug-tolerant persisters and residual disease following targeted therapy, chemotherapy, and radiotherapy. Collectively, our study provides novel targets and mechanistic insights that can guide the development of effective combinatorial targeted therapies, thus maximizing the therapeutic benefits of TKIs.

RevDate: 2025-04-17
CmpDate: 2025-04-17

Zhao X, Mai C, Xia L, et al (2025)

Molecular Insights into the Positive Role of Soybean Nodulation by GmWRKY17.

International journal of molecular sciences, 26(7):.

Soybean is an important economic oilseed crop, being rich in protein and plant oil, it is widely cultivated around the world. Soybeans have been shown to establish a symbiotic nitrogen fixation (SNF) with their compatible rhizobia, resulting in the formation of nodules. Previous studies have demonstrated the critical roles of phytohormones, such as abscisic acid and cytokinin, in the process of legume nodulation. The present study investigated the role of GmWRKY17, a homolog of Rosa hybrida (Rh)WRKY13 in regulating plant immunity through cytokinin content and abscisic acid signaling in soybean nodulation. Utilizing real-time PCR and histochemical staining, we demonstrated that GmWRKY17 is predominantly expressed in soybean root nodules. Subsequently, we analyzed the function of GmWRKY17-overexpression, RNA interference (RNAi), and the CRISPR/Cas9 system. Overexpression of GmWRKY17 significantly increases soybean nodule number, while RNAi or CRISPR/Cas9-mediated knockout of GmWRKY17 resulted in a dramatic repression of nodule formation in soybeans. These results highlight that GmWRKY17 functions as a positive regulator involved in soybean nodulation. Furthermore, manipulation of GmWRKY17 expression impacts the expression of genes associated with the nod factor (NF) signaling pathway, thereby influencing soybean nodulation. This study demonstrated that WRKY-type transcription factors are involved in the regulation of legume nodulation, offering new light on the molecular basis of the symbiotic interaction between legumes and rhizobia.

RevDate: 2025-04-18
CmpDate: 2025-04-18

Li M, Pan L, Ma C, et al (2025)

Tracking of single virus: Dual fluorescent labeling of pseudorabies virus for observing entry and replication in the N2a cells.

Veterinary microbiology, 304:110503.

Pseudorabies virus (PRV) is a neurotropic herpesvirus. It is not easy to be track the whole replication progress of PRV, especially the nascent viral genome in the host cells. In this study, we developed a dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) with the viral genome and the envelope protein gM labeled by ANCHOR DNA labeling system and mCherry, respectively. Through single-virus tracking of rPRV-Anchor3-mCherry, we observed that PRV invaded mouse neuroblastoma Neuro-2a cells via both endocytosis and plasma membrane fusion pathway. During the replication stage, parental and progeny viral genome of rPRV-Anchor3-mCherry in the cell nuclei could be visible, and viral nucleocapsid appeared more specifically than traditional capsid protein labeled PRV particles (rPRV-VP26-EGFP). We found that numerous progeny viral particles were produced in the nuclear, causing the nucleus membrane to break using three-dimensional (3D) live-cell imaging and electron microscopy. Moreover, our findings confirmed that simultaneously targeting of the UL9 and UL54 genes using a CRISPR-Cas9 system led to the complete inhibition PRV replication. rPRV-Anchor3-mCherry can be used to research multiple steps of the viral cycle.

RevDate: 2025-04-18
CmpDate: 2025-04-18

Lee JS, Dan T, Zhang H, et al (2025)

An ultraconserved snoRNA-like element in long noncoding RNA CRNDE promotes ribosome biogenesis and cell proliferation.

Molecular cell, 85(8):1543-1560.e10.

Cancer cells frequently upregulate ribosome production to support tumorigenesis. While small nucleolar RNAs (snoRNAs) are critical for ribosome biogenesis, the roles of other classes of noncoding RNAs in this process remain largely unknown. Here, we performed CRISPR interference (CRISPRi) screens to identify essential long noncoding RNAs (lncRNAs) in renal cell carcinoma (RCC) cells. This revealed that an alternatively spliced isoform of lncRNA colorectal neoplasia differentially expressed (CRNDE) containing an ultraconserved element (UCE), referred to as CRNDE[UCE], is required for RCC cell proliferation. CRNDE[UCE] localizes to the nucleolus and promotes 60S ribosomal subunit biogenesis. The UCE of CRNDE functions as an unprocessed C/D box snoRNA that directly interacts with ribosomal RNA precursors. This facilitates delivery of eukaryotic initiation factor 6 (eIF6), a key 60S biogenesis factor, which binds to CRNDE[UCE] through a sequence element adjacent to the UCE. These findings highlight the functional versatility of snoRNA sequences and expand the known mechanisms through which noncoding RNAs orchestrate ribosome biogenesis.

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ESP Quick Facts

ESP Origins

In the early 1990's, Robert Robbins was a faculty member at Johns Hopkins, where he directed the informatics core of GDB — the human gene-mapping database of the international human genome project. To share papers with colleagues around the world, he set up a small paper-sharing section on his personal web page. This small project evolved into The Electronic Scholarly Publishing Project.

ESP Support

In 1995, Robbins became the VP/IT of the Fred Hutchinson Cancer Research Center in Seattle, WA. Soon after arriving in Seattle, Robbins secured funding, through the ELSI component of the US Human Genome Project, to create the original ESP.ORG web site, with the formal goal of providing free, world-wide access to the literature of classical genetics.

ESP Rationale

Although the methods of molecular biology can seem almost magical to the uninitiated, the original techniques of classical genetics are readily appreciated by one and all: cross individuals that differ in some inherited trait, collect all of the progeny, score their attributes, and propose mechanisms to explain the patterns of inheritance observed.

ESP Goal

In reading the early works of classical genetics, one is drawn, almost inexorably, into ever more complex models, until molecular explanations begin to seem both necessary and natural. At that point, the tools for understanding genome research are at hand. Assisting readers reach this point was the original goal of The Electronic Scholarly Publishing Project.

ESP Usage

Usage of the site grew rapidly and has remained high. Faculty began to use the site for their assigned readings. Other on-line publishers, ranging from The New York Times to Nature referenced ESP materials in their own publications. Nobel laureates (e.g., Joshua Lederberg) regularly used the site and even wrote to suggest changes and improvements.

ESP Content

When the site began, no journals were making their early content available in digital format. As a result, ESP was obliged to digitize classic literature before it could be made available. For many important papers — such as Mendel's original paper or the first genetic map — ESP had to produce entirely new typeset versions of the works, if they were to be available in a high-quality format.

ESP Help

Early support from the DOE component of the Human Genome Project was critically important for getting the ESP project on a firm foundation. Since that funding ended (nearly 20 years ago), the project has been operated as a purely volunteer effort. Anyone wishing to assist in these efforts should send an email to Robbins.

ESP Plans

With the development of methods for adding typeset side notes to PDF files, the ESP project now plans to add annotated versions of some classical papers to its holdings. We also plan to add new reference and pedagogical material. We have already started providing regularly updated, comprehensive bibliographies to the ESP.ORG site.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

Electronic Scholarly Publishing
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Papers in Classical Genetics

The ESP began as an effort to share a handful of key papers from the early days of classical genetics. Now the collection has grown to include hundreds of papers, in full-text format.

Digital Books

Along with papers on classical genetics, ESP offers a collection of full-text digital books, including many works by Darwin and even a collection of poetry — Chicago Poems by Carl Sandburg.

Timelines

ESP now offers a large collection of user-selected side-by-side timelines (e.g., all science vs. all other categories, or arts and culture vs. world history), designed to provide a comparative context for appreciating world events.

Biographies

Biographical information about many key scientists (e.g., Walter Sutton).

Selected Bibliographies

Bibliographies on several topics of potential interest to the ESP community are automatically maintained and generated on the ESP site.

ESP Picks from Around the Web (updated 28 JUL 2024 )