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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 18 Mar 2024 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: 2024-03-13
CmpDate: 2024-03-13

Lee HJ, SJ Lee (2024)

Single-Nucleotide Microbial Genome Editing Using CRISPR-Cas12a.

Methods in molecular biology (Clifton, N.J.), 2760:147-155.

Microbial genome editing can be achieved by donor DNA-directed mutagenesis and CRISPR-Cas12a-mediated negative selection. Single-nucleotide-level genome editing enables the manipulation of microbial cells exactly as designed. Here, we describe single-nucleotide substitutions/indels in the target DNA of E. coli genome using a mutagenic DNA oligonucleotide donor and truncated crRNA/Cas12a system. The maximal truncation of nucleotides at the 3'-end of the crRNA enables Cas12a-mediated single-nucleotide-level precise editing at galK targets in the genome of E. coli.

RevDate: 2024-03-13
CmpDate: 2024-03-13

Kang M, Kim K, BK Cho (2024)

CRISPRi-Driven Genetic Screening for Designing Novel Microbial Phenotypes.

Methods in molecular biology (Clifton, N.J.), 2760:117-132.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has enabled rapid advances in genomic engineering and transcriptional regulation. Specifically, CRISPR interference (CRISPRi) system has been used to systematically investigate the gene functions of microbial strains in a high-throughput manner. This method involves growth profiling using cells that have been transformed with the deactivated Cas9 (dCas9) and single-guide RNA (sgRNA) libraries that target individual genes. The fitness scores of each gene are calculated by measuring the abundance of individual sgRNAs during cell growth and represent gene essentiality. In this chapter, a process is described for functional genetic screening using CRISPRi at the whole-genome scale, starting from the synthesis of sgRNA libraries, construction of CRISPRi libraries, and identification of essential genes through growth profiling. The commensal bacterium Bacteroides thetaiotaomicron was used to implement the protocol. This method is expected to be applicable to a broader range of microorganisms to explore the novel phenotypic characteristics of microorganisms.

RevDate: 2024-03-13
CmpDate: 2024-03-13

Yu L, MA Marchisio (2024)

dCas12a:Pre-crRNA: A New Tool to Induce mRNA Degradation in Saccharomyces cerevisiae Synthetic Gene Circuits.

Methods in molecular biology (Clifton, N.J.), 2760:95-114.

We describe a new way to trigger mRNA degradation in Saccharomyces cerevisiae synthetic gene circuits. Our method demands to modify either the 5'- or the 3'-UTR that flanks a target gene with elements from the pre-crRNA of type V Cas12a proteins and expresses a DNase-deficient Cas12a (dCas12a). dCas12a recognizes and cleaves the pre-crRNA motifs on mRNA sequences. Our tool does not require complex engineering operations and permits an efficient control of protein expression via mRNA degradation.

RevDate: 2024-03-12

O'Leary K (2024)

MEGA CRISPR rejuvenates exhausted CAR T cells.

RevDate: 2024-03-15
CmpDate: 2024-03-13

Kogenaru V, Isalan M, M Kogenaru (2024)

A drug stabilizable GAL80[ds] for conditional control of gene expression via GAL4-UAS and CRISPR-Cas9 systems in Drosophila.

Scientific reports, 14(1):5893.

The binary GAL4-UAS expression system has been widely used in Drosophila to achieve tissue-specific expression of genes. To further allow for simultaneous spatial and conditional control of gene expression in existing GAL4 expression lines backgrounds, temperature and chemical controllable GAL80 variants have been engineered. Here we add a new drug stabilizable GAL80[ds] variant, by fusing it to a low-background DHFR-22-DD. We first quantify both single (DD-GAL80) and double (DD-GAL80-DD) architectures and show varied background and activation levels. Next, we demonstrate the utility of GAL80[ds] Drosophila line to regulate a cell death gene ectopically, in a drug-dependent manner, by utilizing an existing tissue-specific GAL4 driver that regulates the expression of a cell death gene under a UAS. Finally, we showcase the usefulness of GAL80[ds] in tight drug-mediated regulation of a target gene, from an endogenous locus, by utilizing an existing tissue-specific GAL4 to drive the expression of a dead Cas9 variant fused to the transcriptional coactivator nejire, under a UAS and in gRNA lines. Overall, these new GAL80[ds] lines expand the use of the wide variety of existing tissue-specific GAL4 and gene-specific gRNA lines. This enables conditional control of genes, both ectopically and endogenously, for a broad array of gene expression control applications.

RevDate: 2024-03-15
CmpDate: 2024-03-13

Apostolopoulos A, Kawamoto N, Chow SYA, et al (2024)

dCas13-mediated translational repression for accurate gene silencing in mammalian cells.

Nature communications, 15(1):2205.

Current gene silencing tools based on RNA interference (RNAi) or, more recently, clustered regularly interspaced short palindromic repeats (CRISPR)‒Cas13 systems have critical drawbacks, such as off-target effects (RNAi) or collateral mRNA cleavage (CRISPR‒Cas13). Thus, a more specific method of gene knockdown is needed. Here, we develop CRISPRδ, an approach for translational silencing, harnessing catalytically inactive Cas13 proteins (dCas13). Owing to its tight association with mRNA, dCas13 serves as a physical roadblock for scanning ribosomes during translation initiation and does not affect mRNA stability. Guide RNAs covering the start codon lead to the highest efficacy regardless of the translation initiation mechanism: cap-dependent, internal ribosome entry site (IRES)-dependent, or repeat-associated non-AUG (RAN) translation. Strikingly, genome-wide ribosome profiling reveals the ultrahigh gene silencing specificity of CRISPRδ. Moreover, the fusion of a translational repressor to dCas13 further improves the performance. Our method provides a framework for translational repression-based gene silencing in eukaryotes.

RevDate: 2024-03-15
CmpDate: 2024-03-15

Saikia BB, Bhowmick S, Malat A, et al (2024)

ICAM-1 Deletion Using CRISPR/Cas9 Protects the Brain from Traumatic Brain Injury-Induced Inflammatory Leukocyte Adhesion and Transmigration Cascades by Attenuating the Paxillin/FAK-Dependent Rho GTPase Pathway.

The Journal of neuroscience : the official journal of the Society for Neuroscience, 44(11): pii:JNEUROSCI.1742-23.2024.

Intercellular adhesion molecule-1 (ICAM-1) is identified as an initiator of neuroinflammatory responses that lead to neurodegeneration and cognitive and sensory-motor deficits in several pathophysiological conditions including traumatic brain injury (TBI). However, the underlying mechanisms of ICAM-1-mediated leukocyte adhesion and transmigration and its link with neuroinflammation and functional deficits following TBI remain elusive. Here, we hypothesize that blocking of ICAM-1 attenuates the transmigration of leukocytes to the brain and promotes functional recovery after TBI. The experimental TBI was induced in vivo by fluid percussion injury (25 psi) in male and female wild-type and ICAM-1[-/-] mice and in vitro by stretch injury (3 psi) in human brain microvascular endothelial cells (hBMVECs). We treated hBMVECs and animals with ICAM-1 CRISPR/Cas9 and conducted several biochemical analyses and demonstrated that CRISPR/Cas9-mediated ICAM-1 deletion mitigates blood-brain barrier (BBB) damage and leukocyte transmigration to the brain by attenuating the paxillin/focal adhesion kinase (FAK)-dependent Rho GTPase pathway. For analyzing functional outcomes, we used a cohort of behavioral tests that included sensorimotor functions, psychological stress analyses, and spatial memory and learning following TBI. In conclusion, this study could establish the significance of deletion or blocking of ICAM-1 in transforming into a novel preventive approach against the pathophysiology of TBI.

RevDate: 2024-03-13
CmpDate: 2024-03-13

Wilson EL, Yu Y, Leal NS, et al (2024)

Genome-wide CRISPR/Cas9 screen shows that loss of GET4 increases mitochondria-endoplasmic reticulum contact sites and is neuroprotective.

Cell death & disease, 15(3):203.

Organelles form membrane contact sites between each other, allowing for the transfer of molecules and signals. Mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) are cellular subdomains characterized by close apposition of mitochondria and ER membranes. They have been implicated in many diseases, including neurodegenerative, metabolic, and cardiac diseases. Although MERCS have been extensively studied, much remains to be explored. To uncover novel regulators of MERCS, we conducted a genome-wide, flow cytometry-based screen using an engineered MERCS reporter cell line. We found 410 genes whose downregulation promotes MERCS and 230 genes whose downregulation decreases MERCS. From these, 29 genes were selected from each population for arrayed screening and 25 were validated from the high population and 13 from the low population. GET4 and BAG6 were highlighted as the top 2 genes that upon suppression increased MERCS from both the pooled and arrayed screens, and these were subjected to further investigation. Multiple microscopy analyses confirmed that loss of GET4 or BAG6 increased MERCS. GET4 and BAG6 were also observed to interact with the known MERCS proteins, inositol 1,4,5-trisphosphate receptors (IP3R) and glucose-regulated protein 75 (GRP75). In addition, we found that loss of GET4 increased mitochondrial calcium uptake upon ER-Ca[2+] release and mitochondrial respiration. Finally, we show that loss of GET4 rescues motor ability, improves lifespan and prevents neurodegeneration in a Drosophila model of Alzheimer's disease (Aβ42Arc). Together, these results suggest that GET4 is involved in decreasing MERCS and that its loss is neuroprotective.

RevDate: 2024-03-14

Ligunas GD, Paniagua G, LaBelle J, et al (2024)

Tissue-specific and endogenous protein labeling with split fluorescent proteins.

bioRxiv : the preprint server for biology.

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein function. Our approach should be broadly useful for a wide range of applications.

RevDate: 2024-03-12
CmpDate: 2024-03-11

Boudna M, Campos AD, Vychytilova-Faltejskova P, et al (2024)

Strategies for labelling of exogenous and endogenous extracellular vesicles and their application for in vitro and in vivo functional studies.

Cell communication and signaling : CCS, 22(1):171.

This review presents a comprehensive overview of labelling strategies for endogenous and exogenous extracellular vesicles, that can be utilised both in vitro and in vivo. It covers a broad spectrum of approaches, including fluorescent and bioluminescent labelling, and provides an analysis of their applications, strengths, and limitations. Furthermore, this article presents techniques that use radioactive tracers and contrast agents with the ability to track EVs both spatially and temporally. Emphasis is also placed on endogenous labelling mechanisms, represented by Cre-lox and CRISPR-Cas systems, which are powerful and flexible tools for real-time EV monitoring or tracking their fate in target cells. By summarizing the latest developments across these diverse labelling techniques, this review provides researchers with a reference to select the most appropriate labelling method for their EV based research.

RevDate: 2024-03-09

Biase FH, G Schettini (2024)

Protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes.

STAR protocols, 5(1):102940 pii:S2666-1667(24)00105-9 [Epub ahead of print].

The use of CRISPR-Cas9 ribonucleoproteins has revolutionized manipulation of genomes. Here, we present a protocol for the electroporation of CRISPR-Cas for DNA and RNA targeting in Bos taurus zygotes. First, we describe steps for production and preparation of presumptive zygotes for electroporation. The first electroporation introduces ribonucleoproteins formed by Cas9D10A with two guide RNAs to target DNA, and the second introduces the same ribonucleoprotein complex to target DNA plus Cas13a with one guide RNA to target RNAs. For complete details on the use and execution of this protocol, please refer to Nix et al.[1].

RevDate: 2024-03-09

Klermund J, Rhiel M, Kocher T, et al (2024)

On- and off-target effects of paired CRISPR-Cas nickase in primary human cells.

Molecular therapy : the journal of the American Society of Gene Therapy pii:S1525-0016(24)00147-3 [Epub ahead of print].

Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-Seq pipeline, D-CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from epidermolysis bullosa patients. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired nickase editing. While the double nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects, but still leave substantial chromosomal aberrations at on-target sites.

RevDate: 2024-03-12
CmpDate: 2024-03-11

Liu Y, Chao Z, Ding W, et al (2024)

A multiplex RPA-CRISPR/Cas12a-based POCT technique and its application in human papillomavirus (HPV) typing assay.

Cellular & molecular biology letters, 29(1):34.

Persistent infection with high-risk human papillomavirus (HR-HPV) is the primary and initiating factor for cervical cancer. With over 200 identified HPV types, including 14 high-risk types that integrate into the host cervical epithelial cell DNA, early determination of HPV infection type is crucial for effective risk stratification and management. Presently, on-site immediate testing during the HPV screening stage, known as Point of Care Testing (POCT), remains immature, severely limiting the scope and scenarios of HPV screening. This study, guided by the genomic sequence patterns of HPV, established a multiplex recombinase polymerase amplification (RPA) technology based on the concept of "universal primers." This approach achieved the multiple amplification of RPA, coupled with the CRISPR/Cas12a system serving as a medium for signal amplification and conversion. The study successfully constructed a POCT combined detection system, denoted as H-MRC12a (HPV-Multiple RPA-CRISPR/Cas12a), and applied it to high-risk HPV typing detection. The system accomplished the typing detection of six high-risk HPV types (16, 18, 31, 33, 35, and 45) can be completed within 40 min, and the entire process, from sample loading to result interpretation, can be accomplished within 45 min, with a detection depth reaching 1 copy/μL for each high-risk type. Validation of the H-MRC12a detection system's reproducibility and specificity was further conducted through QPCR on 34 clinical samples. Additionally, this study explored and optimized the multiplex RPA amplification system and CRISPR system at the molecular mechanism level. Furthermore, the primer design strategy developed in this study offers the potential to enhance the throughput of H-MRC12a detection while ensuring sensitivity, providing a novel research avenue for high-throughput detection in Point-of-Care molecular pathogen studies.

RevDate: 2024-03-14
CmpDate: 2024-03-14

Han Y, Liu G, Wu Y, et al (2024)

CrisprStitch: Fast evaluation of the efficiency of CRISPR editing systems.

Plant communications, 5(3):100783.

RevDate: 2024-03-14
CmpDate: 2024-03-14

Wang H, Zhang D, Chen M, et al (2024)

Genome editing of 3' UTR-embedded inhibitory region enables generation of gene knock-up alleles in plants.

Plant communications, 5(3):100745.

RevDate: 2024-03-12
CmpDate: 2024-03-11

Beavogui A, Lacroix A, Wiart N, et al (2024)

The defensome of complex bacterial communities.

Nature communications, 15(1):2146.

Bacteria have developed various defense mechanisms to avoid infection and killing in response to the fast evolution and turnover of viruses and other genetic parasites. Such pan-immune system (defensome) encompasses a growing number of defense lines that include well-studied innate and adaptive systems such as restriction-modification, CRISPR-Cas and abortive infection, but also newly found ones whose mechanisms are still poorly understood. While the abundance and distribution of defense systems is well-known in complete and culturable genomes, there is a void in our understanding of their diversity and richness in complex microbial communities. Here we performed a large-scale in-depth analysis of the defensomes of 7759 high-quality bacterial population genomes reconstructed from soil, marine, and human gut environments. We observed a wide variation in the frequency and nature of the defensome among large phyla, which correlated with lifestyle, genome size, habitat, and geographic background. The defensome's genetic mobility, its clustering in defense islands, and genetic variability was found to be system-specific and shaped by the bacterial environment. Hence, our results provide a detailed picture of the multiple immune barriers present in environmentally distinct bacterial communities and set the stage for subsequent identification of novel and ingenious strategies of diversification among uncultivated microbes.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Martinsen E, Jinnurine T, Subramani S, et al (2024)

Advances in RNA therapeutics for modulation of 'undruggable' targets.

Progress in molecular biology and translational science, 204:249-294.

Over the past decades, drug discovery utilizing small pharmacological compounds, fragment-based therapeutics, and antibody therapy have significantly advanced treatment options for many human diseases. However, a major bottleneck has been that>70% of human proteins/genomic regions are 'undruggable' by the above-mentioned approaches. Many of these proteins constitute essential drug targets against complex multifactorial diseases like cancer, immunological disorders, and neurological diseases. Therefore, alternative approaches are required to target these proteins or genomic regions in human cells. RNA therapeutics is a promising approach for many of the traditionally 'undruggable' targets by utilizing methods such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based genome editing, aptamers, and the development of mRNA therapeutics. In the following chapter, we will put emphasis on recent advancements utilizing these approaches against challenging drug targets, such as intranuclear proteins, intrinsically disordered proteins, untranslated genomic regions, and targets expressed in inaccessible tissues.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Li Y, Zhou S, Wu Q, et al (2024)

CRISPR/Cas gene editing and delivery systems for cancer therapy.

Wiley interdisciplinary reviews. Nanomedicine and nanobiotechnology, 16(1):e1938.

CRISPR/Cas systems stand out because of simplicity, efficiency, and other superiorities, thus becoming attractive and brilliant gene-editing tools in biomedical field including cancer therapy. CRISPR/Cas systems bring promises for cancer therapy through manipulating and engineering on tumor cells or immune cells. However, there have been concerns about how to overcome the numerous physiological barriers and deliver CRISPR components to target cells efficiently and accurately. In this review, we introduced the mechanisms of CRISPR/Cas systems, summarized the current delivery strategies of CRISPR/Cas systems by physical methods, viral vectors, and nonviral vectors, and presented the current application of CRISPR/Cas systems in cancer clinical treatment. Furthermore, we discussed prospects related to delivery approaches of CRISPR/Cas systems. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Cai H, Zhang B, Ahrenfeldt J, et al (2024)

CRISPR/Cas9 model of prostate cancer identifies Kmt2c deficiency as a metastatic driver by Odam/Cabs1 gene cluster expression.

Nature communications, 15(1):2088.

Metastatic prostate cancer (PCa) poses a significant therapeutic challenge with high mortality rates. Utilizing CRISPR-Cas9 in vivo, we target five potential tumor suppressor genes (Pten, Trp53, Rb1, Stk11, and RnaseL) in the mouse prostate, reaching humane endpoint after eight weeks without metastasis. By further depleting three epigenetic factors (Kmt2c, Kmt2d, and Zbtb16), lung metastases are present in all mice. While whole genome sequencing reveals few mutations in coding sequence, RNA sequencing shows significant dysregulation, especially in a conserved genomic region at chr5qE1 regulated by KMT2C. Depleting Odam and Cabs1 in this region prevents metastasis. Notably, the gene expression signatures, resulting from our study, predict progression-free and overall survival and distinguish primary and metastatic human prostate cancer. This study emphasizes positive genetic interactions between classical tumor suppressor genes and epigenetic modulators in metastatic PCa progression, offering insights into potential treatments.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Hayashi N, Lai Y, Fuerte-Stone J, et al (2024)

Cas9-assisted biological containment of a genetically engineered human commensal bacterium and genetic elements.

Nature communications, 15(1):2096.

Sophisticated gene circuits built by synthetic biology can enable bacteria to sense their environment and respond predictably. Engineered biosensing bacteria outfitted with such circuits can potentially probe the human gut microbiome to prevent, diagnose, or treat disease. To provide robust biocontainment for engineered bacteria, we devised a Cas9-assisted auxotrophic biocontainment system combining thymidine auxotrophy, an Engineered Riboregulator (ER) for controlled gene expression, and a CRISPR Device (CD). The CD prevents the engineered bacteria from acquiring thyA via horizontal gene transfer, which would disrupt the biocontainment system, and inhibits the spread of genetic elements by killing bacteria harboring the gene cassette. This system tunably controlled gene expression in the human gut commensal bacterium Bacteroides thetaiotaomicron, prevented escape from thymidine auxotrophy, and blocked transgene dissemination. These capabilities were validated in vitro and in vivo. This biocontainment system exemplifies a powerful strategy for bringing genetically engineered microorganisms safely into biomedicine.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Weber Y, Böck D, Ivașcu A, et al (2024)

Enhancing prime editor activity by directed protein evolution in yeast.

Nature communications, 15(1):2092.

Prime editing is a highly versatile genome editing technology that enables the introduction of base substitutions, insertions, and deletions. However, compared to traditional Cas9 nucleases prime editors (PEs) are less active. In this study we use OrthoRep, a yeast-based platform for directed protein evolution, to enhance the editing efficiency of PEs. After several rounds of evolution with increased selection pressure, we identify multiple mutations that have a positive effect on PE activity in yeast cells and in biochemical assays. Combining the two most effective mutations - the A259D amino acid substitution in nCas9 and the K445T substitution in M-MLV RT - results in the variant PE_Y18. Delivery of PE_Y18, encoded on DNA, mRNA or as a ribonucleoprotein complex into mammalian cell lines increases editing rates up to 3.5-fold compared to PEmax. In addition, PE_Y18 supports higher prime editing rates when delivered in vivo into the liver or brain. Our study demonstrates proof-of-concept for the application of OrthoRep to optimize genome editing tools in eukaryotic cells.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Wang X, S Leptihn (2024)

Defense and anti-defense mechanisms of bacteria and bacteriophages.

Journal of Zhejiang University. Science. B, 25(3):181-196.

In the post-antibiotic era, the overuse of antimicrobials has led to a massive increase in antimicrobial resistance, leaving medical doctors few or no treatment options to fight infections caused by superbugs. The use of bacteriophages is a promising alternative to treat infections, supplementing or possibly even replacing antibiotics. Using phages for therapy is possible, since these bacterial viruses can kill bacteria specifically, causing no harm to the normal flora. However, bacteria have developed a multitude of sophisticated and complex ways to resist infection by phages, including abortive infection and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. Phages also can evolve and acquire new anti-defense strategies to continue predation. An in-depth exploration of both defense and anti-defense mechanisms would contribute to optimizing phage therapy, while we would also gain novel insights into the microbial world. In this paper, we summarize recent research on bacterial phage resistance and phage anti-defense mechanisms, as well as collaborative win-win systems involving both virus and host.

RevDate: 2024-03-08

Das S, Kwon M, JY Kim (2024)

Enhancement of specialized metabolites using CRISPR/Cas gene editing technology in medicinal plants.

Frontiers in plant science, 15:1279738.

Plants are the richest source of specialized metabolites. The specialized metabolites offer a variety of physiological benefits and many adaptive evolutionary advantages and frequently linked to plant defense mechanisms. Medicinal plants are a vital source of nutrition and active pharmaceutical agents. The production of valuable specialized metabolites and bioactive compounds has increased with the improvement of transgenic techniques like gene silencing and gene overexpression. These techniques are beneficial for decreasing production costs and increasing nutritional value. Utilizing biotechnological applications to enhance specialized metabolites in medicinal plants needs characterization and identification of genes within an elucidated pathway. The breakthrough and advancement of CRISPR/Cas-based gene editing in improving the production of specific metabolites in medicinal plants have gained significant importance in contemporary times. This article imparts a comprehensive recapitulation of the latest advancements made in the implementation of CRISPR-gene editing techniques for the purpose of augmenting specific metabolites in medicinal plants. We also provide further insights and perspectives for improving metabolic engineering scenarios in medicinal plants.

RevDate: 2024-03-08
CmpDate: 2024-03-08

Tao R, Han X, Bai X, et al (2024)

Revolutionizing cancer treatment: enhancing CAR-T cell therapy with CRISPR/Cas9 gene editing technology.

Frontiers in immunology, 15:1354825.

CAR-T cell therapy, a novel immunotherapy, has made significant breakthroughs in clinical practice, particularly in treating B-cell-associated leukemia and lymphoma. However, it still faces challenges such as poor persistence, limited proliferation capacity, high manufacturing costs, and suboptimal efficacy. CRISPR/Cas system, an efficient and simple method for precise gene editing, offers new possibilities for optimizing CAR-T cells. It can increase the function of CAR-T cells and reduce manufacturing costs. The combination of CRISPR/Cas9 technology and CAR-T cell therapy may promote the development of this therapy and provide more effective and personalized treatment for cancer patients. Meanwhile, the safety issues surrounding the application of this technology in CAR-T cells require further research and evaluation. Future research should focus on improving the accuracy and safety of CRISPR/Cas9 technology to facilitate the better development and application of CAR-T cell therapy. This review focuses on the application of CRISPR/Cas9 technology in CAR-T cell therapy, including eliminating the inhibitory effect of immune checkpoints, enhancing the ability of CAR-T cells to resist exhaustion, assisting in the construction of universal CAR-T cells, reducing the manufacturing costs of CAR-T cells, and the security problems faced. The objective is to show the revolutionary role of CRISPR/Cas9 technology in CAR-T cell therapy for researchers.

RevDate: 2024-03-08
CmpDate: 2024-03-08

Mori K, Tanase K, K Sasaki (2024)

Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

Planta, 259(4):84.

A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

RevDate: 2024-03-09
CmpDate: 2024-03-08

Uppuluri L, Shi CH, Varapula D, et al (2024)

A long-read sequencing strategy with overlapping linkers on adjacent fragments (OLAF-Seq) for targeted resequencing and enrichment.

Scientific reports, 14(1):5583.

In this report, we present OLAF-Seq, a novel strategy to construct a long-read sequencing library such that adjacent fragments are linked with end-terminal duplications. We use the CRISPR-Cas9 nickase enzyme and a pool of multiple sgRNAs to perform non-random fragmentation of targeted long DNA molecules (> 300kb) into smaller library-sized fragments (about 20 kbp) in a manner so as to retain physical linkage information (up to 1000 bp) between adjacent fragments. DNA molecules targeted for fragmentation are preferentially ligated with adaptors for sequencing, so this method can enrich targeted regions while taking advantage of the long-read sequencing platforms. This enables the sequencing of target regions with significantly lower total coverage, and the genome sequence within linker regions provides information for assembly and phasing. We demonstrated the validity and efficacy of the method first using phage and then by sequencing a panel of 100 full-length cancer-related genes (including both exons and introns) in the human genome. When the designed linkers contained heterozygous genetic variants, long haplotypes could be established. This sequencing strategy can be readily applied in both PacBio and Oxford Nanopore platforms for both long and short genes with an easy protocol. This economically viable approach is useful for targeted enrichment of hundreds of target genomic regions and where long no-gap contigs need deep sequencing.

RevDate: 2024-03-13
CmpDate: 2024-03-08

Wong CH, Wingett SW, Qian C, et al (2024)

Genome-scale requirements for dynein-based transport revealed by a high-content arrayed CRISPR screen.

The Journal of cell biology, 223(5):.

The microtubule motor dynein plays a key role in cellular organization. However, little is known about how dynein's biosynthesis, assembly, and functional diversity are orchestrated. To address this issue, we have conducted an arrayed CRISPR loss-of-function screen in human cells using the distribution of dynein-tethered peroxisomes and early endosomes as readouts. From a genome-wide gRNA library, 195 validated hits were recovered and parsed into those impacting multiple dynein cargoes and those whose effects are restricted to a subset of cargoes. Clustering of high-dimensional phenotypic fingerprints revealed co-functional proteins involved in many cellular processes, including several candidate novel regulators of core dynein functions. Further analysis of one of these factors, the RNA-binding protein SUGP1, indicates that it promotes cargo trafficking by sustaining functional expression of the dynein activator LIS1. Our data represent a rich source of new hypotheses for investigating microtubule-based transport, as well as several other aspects of cellular organization captured by our high-content imaging.

RevDate: 2024-03-08
CmpDate: 2024-03-08

Mathur S, Singh D, R Ranjan (2024)

Recent advances in plant translational genomics for crop improvement.

Advances in protein chemistry and structural biology, 139:335-382.

The growing population, climate change, and limited agricultural resources put enormous pressure on agricultural systems. A plateau in crop yields is occurring and extreme weather events and urbanization threaten the livelihood of farmers. It is imperative that immediate attention is paid to addressing the increasing food demand, ensuring resilience against emerging threats, and meeting the demand for more nutritious, safer food. Under uncertain conditions, it is essential to expand genetic diversity and discover novel crop varieties or variations to develop higher and more stable yields. Genomics plays a significant role in developing abundant and nutrient-dense food crops. An alternative to traditional breeding approach, translational genomics is able to improve breeding programs in a more efficient and precise manner by translating genomic concepts into practical tools. Crop breeding based on genomics offers potential solutions to overcome the limitations of conventional breeding methods, including improved crop varieties that provide more nutritional value and are protected from biotic and abiotic stresses. Genetic markers, such as SNPs and ESTs, contribute to the discovery of QTLs controlling agronomic traits and stress tolerance. In order to meet the growing demand for food, there is a need to incorporate QTLs into breeding programs using marker-assisted selection/breeding and transgenic technologies. This chapter primarily focuses on the recent advances that are made in translational genomics for crop improvement and various omics techniques including transcriptomics, metagenomics, pangenomics, single cell omics etc. Numerous genome editing techniques including CRISPR Cas technology and their applications in crop improvement had been discussed.

RevDate: 2024-03-06

Pasch T, Bäumer N, Bäumer S, et al (2024)

Towards targeted Cas9 (CRISPR-Cas) delivery: Preparation of IgG antibody-Cas9 conjugates using a split intein.

Journal of peptide science : an official publication of the European Peptide Society [Epub ahead of print].

The CRISPR-Cas9 system has revolutionized the field of genetic engineering, but targeted cellular delivery remains a central problem. The delivery of the preformed ribonuclease-protein (RNP) complex has the advantages of fewer side effects and avoidance of potential permanent effects. We reasoned that an internalizing IgG antibody as a targeting device could address the delivery of Cas9-RNP. We opted for protein trans-splicing mediated by a split intein to facilitate posttranslational conjugation of the two large protein entities. We recently described the cysteine-less CL split intein that efficiently performs under oxidizing conditions and does not interfere with disulfide bonds or thiol bioconjugation chemistries. Using the CL split intein, we report for the first time the ligation of monoclonal IgG antibody precursors, expressed in mammalian cells, and a Cas9 precursor, obtained from bacterial expression. A purified IgG-Cas9 conjugate was loaded with sgRNA to form the active RNP complex and introduced a double-strand break in its target DNA in vitro. Furthermore, a synthetic peptide variant of the short N-terminal split intein precursor proved useful for chemical modification of Cas9. The split intein ligation procedure reported here for IgG-Cas9 provides the first step towards a novel CRISPR-Cas9 targeting approach involving the preformed RNP complex.

RevDate: 2024-03-13
CmpDate: 2024-03-13

Zhou M, Li C, Wei R, et al (2024)

Exponential Amplification-Induced Activation of CRISPR/Cas9 for Sensitive Detection of Exosomal miRNA.

Analytical chemistry, 96(10):4322-4329.

As an important component of highly heterogeneous exosomes, exosomal microRNAs (miRNAs) have great potential as noninvasive biomarkers for cancer diagnosis. Therefore, a sensitive and simple sensor is the key for its clinical application. Herein, we designed an exponential amplification reaction (EXPAR) to induce the reactivation of the CRISPR-associated protein 9/small guide RNA (Cas9/sgRNA) complex, thus achieving sensitive and visual exosomal miRNAs-21 (miR-21) fluorescence sensing. In this design, we inactivated the sgRNA by hybridizing sgRNA and blocker DNA. Then, we used a trigger DNA to hybridize with miR-21 and produced a lot of activated DNA by EXPAR. Those activated DNA further hybridized with blocker DNA and released the free sgRNA to form the activated Cas9/sgRNA complex. Based on the quick cleavage of activated Cas9/sgRNA complex, the reporter DNA labeled by SYBR Green I was released from the surface of the magnetic nanoparticles (MNPs) into the supernatant, and thus was used to sensitively quantify the miRNAs concentration with a limit of detection of 3 × 10[3] particles/mL. In addition, this fluorescence sensor has also been successfully employed to distinguish healthy people and cancer patients by naked-eye observation of the fluorescence, thus demonstrating its great potential for accurate and point-of-care cancer diagnosis.

RevDate: 2024-03-13
CmpDate: 2024-03-13

Wang ZY, Teng SQ, Zhao NN, et al (2024)

Ligase detection reaction amplification-activated CRISPR-Cas12a for single-molecule counting of FEN1 in breast cancer tissues.

Chemical communications (Cambridge, England), 60(22):3075-3078.

We construct a simple fluorescent biosensor for single-molecule counting of flap endonuclease 1 (FEN1) based on ligase detection reaction (LDR) amplification-activated CRISPR-Cas12a. This biosensor exhibits excellent selectivity and high sensitivity with a detection limit (LOD) of 1.31 × 10[-8] U. Moreover, it can be employed to screen the FEN1 inhibitors and quantitatively measure the FEN1 activity in human cells and breast cancer tissues, holding great promise in clinical diagnosis and drug discovery.

RevDate: 2024-03-13
CmpDate: 2024-03-13

Truong DJ, Geilenkeuser J, Wendel SV, et al (2024)

Exonuclease-enhanced prime editors.

Nature methods, 21(3):455-464.

Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.

RevDate: 2024-03-13
CmpDate: 2024-03-13

Qu Z, Gong Z, Olajide JS, et al (2024)

CRISPR-Cas9-based method for isolating microgametes of Eimeria tenella.

Veterinary parasitology, 327:110131.

Eimeria tenella infections are known to cause severe caecal damage and death of the infected chicken. Gamogony is an essential stage in E. tenella life cycle and in the establishment of coccidiosis. Prior research had extensively explored isolation and separation of the parasite gametes - microgamete (male) and macrogamete (female). However, there is little information on the efficient, highly purified and distinctly separated male and female gametes. In this study, we generated a genome editing line expressing mCherry fluorescent protein fused with GCS1 protein in E. tenella by using Toxoplasma gondii CRISPR-Cas9 system, flow cytometry and fluorescence microscopy. This allowed precise separation of E. tenella male and female gametes in the transgenic parasite population. The separation of male and female gametes would not only build on our understanding of E. tenella transmission, but it would also facilitate development of gametocidal compounds as drug targets for E. tenella infection.

RevDate: 2024-03-13
CmpDate: 2024-03-13

Wu M, Wu H, Chen X, et al (2024)

RPA-CRISPR/Cas9-based method for the detection of Toxoplasma gondii: A proof of concept.

Veterinary parasitology, 327:110115.

Toxoplasma gondii is a widespread and specialized intracellular protozoan pathogen that affects one third of the world' s population, posing a great threat to public health. As the definitive host, cats excrete oocysts and play a crucial role in the transmission of toxoplasmosis. The current diagnostic tools usually require bulky equipment and expertize, which hinders the efficient diagnosis and intervention of Toxoplasma infection in cats. In this study, we combined (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to establish an easier method for the detection of T. gondii oocysts in cat fecal samples. The sensitivity, specificity, and practicability of the established RPA-CRISPR/Cas9 method were evaluated using a lateral flow strip, with the limitation of detection determined at 10 plasmid copies/μL (corresponding to about one oocyst), cross reactivity to none of Giardia lamblia, Cryptosporidium sp., Microsporidium biberi and Blastocystis hominis that also commonly found in cats, and comparable performance in detecting T. gondii in clinical samples to conventional PCR amplification. This RPA-CRISPR/Cas9 method provides an alternative to conventional molecular tools used in the clinical diagnosis of Toxoplasma infection in cats and other animals.

RevDate: 2024-03-09
CmpDate: 2024-03-08

Skeens E, Sinha S, Ahsan M, et al (2024)

High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9.

Science advances, 10(10):eadl1045.

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

RevDate: 2024-03-07
CmpDate: 2024-03-07

Maurya VK, Sanjeevi M, Rahul CN, et al (2024)

Finding identical sequence repeats in multiple protein sequences: An algorithm.

Journal of biosciences, 49:.

In recent years, several experimental evidences suggest that amino acid repeats are closely linked to many disease conditions, as they have a significant role in evolution of disordered regions of the polypeptide segments. Even though many algorithms and databases were developed for such analysis, each algorithm has some caveats, like limitation on the number of amino acids within the repeat patterns and number of query protein sequences. To this end, in the present work, a new method called the internal sequence repeats across multiple protein sequences (ISRMPS) is proposed for the first time to identify identical repeats across multiple protein sequences. It also identifies distantly located repeat patterns in various protein sequences. Our method can be applied to study evolutionary relationships, epitope mapping, CRISPR-Cas sequencing methods, and other comparative analytical assessments of protein sequences.

RevDate: 2024-03-08
CmpDate: 2024-03-07

Tomasi F, Pozzi M, M Lauria (2024)

Investigating the mechanisms underlying resistance to chemoterapy and to CRISPR-Cas9 in cancer cell lines.

Scientific reports, 14(1):5402.

Cancer is one of the major causes of death worldwide and the development of multidrug resistance (MDR) in cancer cells is the principal cause of chemotherapy failure. To gain insights into the specific mechanisms of MDR in cancer cell lines, we developed a novel method for the combined analysis of recently published datasets on drug sensitivity and CRISPR loss-of-function screens for the same set of cancer cell lines. For our analysis, we first selected cell lines that consistently exhibit drug resistance across several classes of compounds. We then identified putative resistance genes for each class of compound and used inferred gene regulatory networks (GRNs) to study possible mechanisms underlying the development of MDR in the identified cancer cell lines. We show that the same method of analysis can also be used to identify cell lines that consistently exhibit resistance to the gene knockout effect of the CRISPR-Cas9 technique and to study the possible underlying mechanisms. In the GRN associated to the drug resistant cell lines, we identify genes previously associated with resistance (UHMK1, RALYL, MGST3, USP9X, and ESRG), genes for which an indirect association can be identified (SPINK13, LINC00664, MRPL38, and EMILIN3), and genes that are found to be overexpressed in non-resistant cancer cell lines (MRPL38, EMILIN3 and RALYL). In the GRNs associated to the CRISPR-Cas9 resistance mechanism, none of the identified genes has been previously reported in the admittedly sparse literature on the subject. However, some of these genes have a common role: APBB2, RUNX1T1, ZBTB7C, and ISX regulate transcription, while APBB2, BTG3, ZBTB7C, SZRD1 and LEF1 have a function in regulating proliferation, suggesting a role for these two pathways. While our results are specific for the lung cancer cell lines we selected for this work, our method of analysis can be applied to cell lines from other tissues and for which the required data is available.

RevDate: 2024-03-08
CmpDate: 2024-03-07

Deng F, Li Y, Yang B, et al (2024)

Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis and transforms DNA/RNA sensing.

Nature communications, 15(1):1818.

Control of CRISPR/Cas12a trans-cleavage is crucial for biosensor development. Here, we show that small circular DNA nanostructures which partially match guide RNA sequences only minimally activate Cas12a ribonucleoproteins. However, linearizing these structures restores activation. Building on this finding, an Autocatalytic Cas12a Circular DNA Amplification Reaction (AutoCAR) system is established which allows a single nucleic acid target to activate multiple ribonucleoproteins, and greatly increases the achievable reporter cleavage rates per target. A rate-equation-based model explains the observed near-exponential rate trends. Autocatalysis is also sustained with DNA nanostructures modified with fluorophore-quencher pairs achieving 1 aM level (<1 copy/μL) DNA detection (10[6] times improvement), without additional amplification, within 15 min, at room temperature. The detection range is tuneable, spanning 3 to 11 orders of magnitude. We demonstrate 1 aM level detection of SNP mutations in circulating tumor DNA from blood plasma, genomic DNA (H. Pylori) and RNA (SARS-CoV-2) without reverse transcription as well as colorimetric lateral flow tests of cancer mutations with ~100 aM sensitivity.

RevDate: 2024-03-05

Chang CW, Truong VA, Pham NN, et al (2024)

RNA-guided genome engineering: paradigm shift towards transposons.

Trends in biotechnology pii:S0167-7799(24)00035-0 [Epub ahead of print].

CRISPR-Cas systems revolutionized the genome engineering field but need to induce double-strand breaks (DSBs) and may be difficult to deliver due to their large protein size. Tn7-like transposons such as CRISPR-associated transposons (CASTs) can be repurposed for RNA-guided DSB-free integration, and obligate mobile element guided activity (OMEGA) proteins of the IS200/IS605 transposon family have been developed as hypercompact RNA-guided genome editing tools. CASTs and OMEGA are exciting, innovative genome engineering tools that can improve the precision and efficiency of editing. This review explores the recent developments and uses of CASTs and OMEGA in genome editing across prokaryotic and eukaryotic cells. The pros and cons of these transposon-based systems are deliberated in comparison to other CRISPR systems.

RevDate: 2024-03-06
CmpDate: 2024-03-06

Hwarari D, Radani Y, Ke Y, et al (2024)

CRISPR/Cas genome editing in plants: mechanisms, applications, and overcoming bottlenecks.

Functional & integrative genomics, 24(2):50.

The CRISPR/Cas systems have emerged as transformative tools for precisely manipulating plant genomes and enhancement. It has provided unparalleled applications from modifying the plant genomes to resistant enhancement. This review manuscript summarises the mechanism, application, and current challenges in the CRISPR/Cas genome editing technology. It addresses the molecular mechanisms of different Cas genes, elucidating their applications in various plants through crop improvement, disease resistance, and trait improvement. The advent of the CRISPR/Cas systems has enabled researchers to precisely modify plant genomes through gene knockouts, knock-ins, and gene expression modulation. Despite these successes, the CRISPR/Cas technology faces challenges, including off-target effects, Cas toxicity, and efficiency. In this manuscript, we also discuss these challenges and outline ongoing strategies employed to overcome these challenges, including the development of novel CRISPR/Cas variants with improved specificity and specific delivery methods for different plant species. The manuscript will conclude by addressing the future perspectives of the CRISPR/Cas technology in plants. Although this review manuscript is not conclusive, it aims to provide immense insights into the current state and future potential of CRISPR/Cas in sustainable and secure plant production.

RevDate: 2024-03-12
CmpDate: 2024-03-12

Chrzanowski S, R Batra (2024)

CRISPR-Based Gene Editing Techniques in Pediatric Neurological Disorders.

Pediatric neurology, 153:166-174.

The emergence of gene editing technologies offers a unique opportunity to develop mutation-specific treatments for pediatric neurological disorders. Gene editing systems can potentially alter disease trajectory by correcting dysfunctional mutations or therapeutically altering gene expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches are attractive gene therapy platforms to personalize treatments because of their specificity, ease of design, versatility, and cost. However, many such approaches remain in the early stages of development, with ongoing efforts to optimize editing efficiency, minimize unintended off-target effects, and mitigate pathologic immune responses. Given the rapid evolution of CRISPR-based therapies, it is prudent for the clinically based child neurologist to have a conceptual understanding of what such therapies may entail, including both benefits and risks and how such therapies may be clinically applied. In this review, we describe the fundamentals of CRISPR-based therapies, discuss the opportunities and challenges that have arisen, and highlight preclinical work in several pediatric neurological diseases.

RevDate: 2024-03-12
CmpDate: 2024-03-12

Salomonsson SE, CD Clelland (2024)

Building CRISPR Gene Therapies for the Central Nervous System: A Review.

JAMA neurology, 81(3):283-290.

IMPORTANCE: Gene editing using clustered regularly interspaced short palindromic repeats (CRISPR) holds the promise to arrest or cure monogenic disease if it can be determined which genetic change to create without inducing unintended cellular dysfunction and how to deliver this technology to the target organ reliably and safely. Clinical trials for blood and liver disorders, for which delivery of CRISPR is not limiting, show promise, yet no trials have begun for central nervous system (CNS) indications.

OBSERVATIONS: The CNS is arguably the most challenging target given its innate exclusion of large molecules and its defenses against bacterial invasion (from which CRISPR originates). Herein, the types of CRISPR editing (DNA cutting, base editing, and templated repair) and how these are applied to different genetic variants are summarized. The challenges of delivering genome editors to the CNS, including the viral and nonviral delivery vehicles that may ultimately circumvent these challenges, are discussed. Also, ways to minimize the potential in vivo genotoxic effects of genome editors through delivery vehicle design and preclinical off-target testing are considered. The ethical considerations of germline editing, a potential off-target outcome of any gene editing therapy, are explored. The unique regulatory challenges of a human-specific therapy that cannot be derisked solely in animal models are also discussed.

CONCLUSIONS AND RELEVANCE: An understanding of both the potential benefits and challenges of CRISPR gene therapy better informs the scientific, clinical, regulatory, and timeline considerations of developing CRISPR gene therapy for neurologic diseases.

RevDate: 2024-03-06
CmpDate: 2024-03-06

Gebhardt CM, D Niopek (2024)

Anti-CRISPR Proteins and Their Application to Control CRISPR Effectors in Mammalian Systems.

Methods in molecular biology (Clifton, N.J.), 2774:205-231.

CRISPR-Cas effectors are powerful tools for genome and transcriptome targeting and editing. Naturally, these protein-RNA complexes are part of the microbial innate immune system, which emerged from the evolutionary arms race between microbes and phages. This coevolution has also given rise to so-called anti-CRISPR (Acr) proteins that counteract the CRISPR-Cas adaptive immunity. Acrs constitutively block cognate CRISPR-Cas effectors, e.g., by interfering with guide RNA binding, target DNA/RNA recognition, or target cleavage. In addition to their important role in microbiology and evolution, Acrs have recently gained particular attention for being useful tools and switches to regulate or fine-tune the activity of CRISPR-Cas effectors. Due to their commonly small size, high inhibition potency, and structural and mechanistic versatility, Acrs offer a wide range of potential applications for controlling CRISPR effectors in heterologous systems, including mammalian cells.Here, we review the diverse applications of Acrs in mammalian cells and organisms and discuss the underlying engineering strategies. These applications include (i) persistent blockage of CRISPR-Cas function to create write-protected cells, (ii) reduction of CRISPR-Cas off-target editing, (iii) focusing CRISPR-Cas activity to specific cell types and tissues, (iv) spatiotemporal control of CRISPR effectors based on engineered, opto-, or chemogenetic Acrs, and (v) the use of Acrs for selective binding and detection of CRISPR-Cas effectors in complex samples. We will also highlight potential future applications of Acrs in a biomedical context and point out present challenges that need to be overcome on the way.

RevDate: 2024-03-06
CmpDate: 2024-03-06

Hirosawa M, H Saito (2024)

RNA Switches Using Cas Proteins.

Methods in molecular biology (Clifton, N.J.), 2774:177-192.

Expanding the number of available RNA-binding proteins (RBPs) is vital to establishing posttranscriptional circuits in mammalian cells. We focused on CRISPR-Cas systems and exploited Cas proteins for their versatility as RBPs. The translation of genes encoded in an mRNA becomes regulatable by a Cas protein by inserting a crRNA/sgRNA sequence recognizable by the specific Cas protein into its 5'UTR. These Cas protein-responsive switches vastly expand the available tools in synthetic biology because of the wide range of Cas protein orthologs that can be used as trigger proteins.Here, we describe the design principle of Cas protein-responsive switches, both plasmid and RNA versions, using Streptococcus pyogenes Cas9 (SpCas9) as an example and show an example of its use in mammalian cells, HEK293FT cells.

RevDate: 2024-03-06
CmpDate: 2024-03-06

Yarra R, PJ Krysan (2024)

An SpG-Cas9-based cytosine base editor expands the scope of genome editing in carrot plants.

Plant cell reports, 43(3):82.

SpG Cas9 significantly expands the genome editing scope in carrot with NGN PAM recognition.

RevDate: 2024-03-05

Liu M, Ding Y, Ye Q, et al (2024)

Cold-tolerance mechanisms in foodborne pathogens: Escherichia coli and Listeria monocytogenes as examples.

Critical reviews in food science and nutrition [Epub ahead of print].

The cold chain is an integral part of the modern food industry. Low temperatures can effectively alleviate food loss and the transmission of foodborne diseases caused by microbial reproduction. However, recent reports have highlighted shortcomings in the current cold chain technology's ability to prevent and control cold-tolerant foodborne pathogens. Furthermore, it has been observed that certain cold-chain foods have emerged as new sources of infection for foodborne disease outbreaks. Consequently, there is a pressing need to enhance control measures targeting cold-tolerant pathogens within the existing cold chain system. This paper aims to review the recent advancements in understanding the cold tolerance mechanisms of key model organisms, identify key issues in current research, and explore the potential of utilizing big data and omics technology in future studies.

RevDate: 2024-03-05

Li Y, Ma R, Gao C, et al (2024)

Integrated bacterial transcriptome and host metabolome analysis reveals insights into "Candidatus Liberibacter asiaticus" population dynamics in the fruit pith of three citrus cultivars with different tolerance.

Microbiology spectrum [Epub ahead of print].

"Candidatus Liberibacter asiaticus" (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars ("Shatangju" mandarin, "Guanxi" pomelo, and "Shatian" pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth.IMPORTANCECitrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, "Candidatus Liberibacter asiaticus" (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

RevDate: 2024-03-06

Nadeem S, Riaz Ahmed S, Luqman T, et al (2024)

A comprehensive review on Gossypium hirsutum resistance against cotton leaf curl virus.

Frontiers in genetics, 15:1306469.

Cotton (Gossypium hirsutum L.) is a significant fiber crop. Being a major contributor to the textile industry requires continuous care and attention. Cotton is subjected to various biotic and abiotic constraints. Among these, biotic factors including cotton leaf curl virus (CLCuV) are dominant. CLCuV is a notorious disease of cotton and is acquired, carried, and transmitted by the whitefly (Bemisia tabaci). A cotton plant affected with CLCuV may show a wide range of symptoms such as yellowing of leaves, thickening of veins, upward or downward curling, formation of enations, and stunted growth. Though there are many efforts to protect the crop from CLCuV, long-term results are not yet obtained as CLCuV strains are capable of mutating and overcoming plant resistance. However, systemic-induced resistance using a gene-based approach remained effective until new virulent strains of CLCuV (like Cotton Leaf Curl Burewala Virus and others) came into existence. Disease control by biological means and the development of CLCuV-resistant cotton varieties are in progress. In this review, we first discussed in detail the evolution of cotton and CLCuV strains, the transmission mechanism of CLCuV, the genetic architecture of CLCuV vectors, and the use of pathogen and nonpathogen-based approaches to control CLCuD. Next, we delineate the uses of cutting-edge technologies like genome editing (with a special focus on CRISPR-Cas), next-generation technologies, and their application in cotton genomics and speed breeding to develop CLCuD resistant cotton germplasm in a short time. Finally, we delve into the current obstacles related to cotton genome editing and explore forthcoming pathways for enhancing precision in genome editing through the utilization of advanced genome editing technologies. These endeavors aim to enhance cotton's resilience against CLCuD.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Alhazza A, Mahdipoor P, Hall R, et al (2024)

Modifying peptide/lipid-associated nucleic acids (PLANAs) for CRISPR/Cas9 ribonucleoprotein delivery.

European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 195:106708.

With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50 % replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Gelsinger DR, Vo PLH, Klompe SE, et al (2024)

Bacterial genome engineering using CRISPR-associated transposases.

Nature protocols, 19(3):752-790.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposases have the potential to transform the technology landscape for kilobase-scale genome engineering, by virtue of their ability to integrate large genetic payloads with high accuracy, easy programmability and no requirement for homologous recombination machinery. These transposons encode efficient, CRISPR RNA-guided transposases that execute genomic insertions in Escherichia coli at efficiencies approaching ~100%. Moreover, they generate multiplexed edits when programmed with multiple guides, and function robustly in diverse Gram-negative bacterial species. Here we present a detailed protocol for engineering bacterial genomes using CRISPR-associated transposase (CAST) systems, including guidelines on the available vectors, customization of guide RNAs and DNA payloads, selection of common delivery methods, and genotypic analysis of integration events. We further describe a computational CRISPR RNA design algorithm to avoid potential off-targets, and a CRISPR array cloning pipeline for performing multiplexed DNA insertions. The method presented here allows the isolation of clonal strains containing a novel genomic integration event of interest within 1-2 weeks using available plasmid constructs and standard molecular biology techniques.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Salem AR, Bryant WB, Doja J, et al (2024)

Prime editing in mice with an engineered pegRNA.

Vascular pharmacology, 154:107269.

CRISPR editing involves double-strand breaks in DNA with attending insertions/deletions (indels) that may result in embryonic lethality in mice. The prime editing (PE) platform uses a prime editing guide RNA (pegRNA) and a Cas9 nickase fused to a modified reverse transcriptase to precisely introduce nucleotide substitutions or small indels without the unintended editing associated with DNA double-strand breaks. Recently, engineered pegRNAs (epegRNAs), with a 3'-extension that shields the primer-binding site of the pegRNA from nucleolytic attack, demonstrated superior activity over conventional pegRNAs in cultured cells. Here, we show the inability of three-component CRISPR or conventional PE to incorporate a nonsynonymous substitution in the Capn2 gene, expected to disrupt a phosphorylation site (S50A) in CAPN2. In contrast, an epegRNA with the same protospacer correctly installed the desired edit in two founder mice, as evidenced by robust genotyping assays for the detection of subtle nucleotide substitutions. Long-read sequencing demonstrated sequence fidelity around the edited site as well as top-ranked distal off-target sites. Western blotting and histological analysis of lipopolysaccharide-treated lung tissue revealed a decrease in phosphorylation of CAPN2 and notable alleviation of inflammation, respectively. These results demonstrate the first successful use of an epegRNA for germline transmission in an animal model and provide a solution to targeting essential developmental genes that otherwise may be challenging to edit.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Zhu Y, Tahara SM, Tsukamoto H, et al (2023)

Protocol for generation of humanized HCC mouse model and cancer-driver mutations using CRISPR-Cas9.

STAR protocols, 4(4):102389.

We detail procedures for generating a humanized mouse model of hepatocellular carcinoma (HCC) recapitulating genetic mutations associated with metabolic liver diseases (MLD). We humanized liver parenchymal, non-parenchymal, and hematopoietic cells. We employed CRISPR-Cas9-based ARID1A knockout and constitutively active CTNNB1 knockin combined with an alcohol Western diet to generate cancer-driver mutations commonly found in MLD-HCC patients. This HCC model facilitates the study of tumor-promoting gene-environment interactions. For complete details on the use and execution of this protocol, please refer to Yeh et al.[1].

RevDate: 2024-03-11
CmpDate: 2024-03-11

Yang X, Duan S, Li Z, et al (2023)

Protocol of CRISPR-Cas9 knockout screens for identifying ferroptosis regulators.

STAR protocols, 4(4):102762.

Ferroptosis, an iron-dependent programmed cell death triggered by excessive lipid peroxidation, has shown promising therapeutic potentials in human diseases. Here, we describe a protocol of a CRISPR-Cas9 loss-of-function screen to identify regulators in response to different inducers of ferroptosis. We emphasize the steps of library amplification, drug treatment, high-throughput sequencing preparation, and bioinformatics analysis using model-based analysis of genome-wide CRISPR-Cas9 knockout (MAGeCK). We also present a method to discover the regulators of ferroptosis and verify the potential targets efficiently. For complete details on use and execution of this protocol, please refer to Yang et al. (2023).[1].

RevDate: 2024-03-11
CmpDate: 2024-03-11

Luo J, Lu C, Wang M, et al (2023)

Protocol for generating mutant zebrafish using CRISPR-Cas9 followed by quantitative evaluation of vascular formation.

STAR protocols, 4(4):102753.

The use of vascular-specific transgenic zebrafish provides advantages for identifying new mutations affecting angiogenesis and vascular development. Here, we present a protocol for establishing, screening, and phenotyping CRISPR-Cas9-based mutagenesis in fluorescently labeled transgenic zebrafish. We describe steps for designing single-guide RNA (sgRNA) oligos, synthesizing sgRNA and Cas9 mRNA, and microinjection and generation of mutant lines. We then detail procedures for visualizing dynamic vasculature and quantitatively evaluating vascular formation in transgenic zebrafish. For complete details on the use and execution of this protocol, please refer to Luo et al.[1].

RevDate: 2024-03-11
CmpDate: 2024-03-11

Wang P, Cao Z, Wang Q, et al (2023)

Protocol for CRISPR-Cas9-mediated genome editing to study spermatogenesis in Caenorhabditis elegans.

STAR protocols, 4(4):102720.

Gene silencing by P-element-induced wimpy testis-interacting RNAs is a mechanism to maintain genome integrity in germ cells. Here, we present a protocol for knockin or knockout editing of male germline genome mediated by CRISPR-Cas9 technology in Caenorhabditis elegans. We describe steps for constructing edited plasmids, microinjecting worms with these plasmids, and screening edited worms. We then detail procedures for dissecting released sperm and their observation with fluorescence microscopy. Engineered worms provide a model for studying hermaphrodite/male fertility or protein localization in vivo. For complete details on the use and execution of this protocol, please refer to Wang et al. (2021).[1].

RevDate: 2024-03-11
CmpDate: 2024-03-11

Wu W, Yin Y, Huang J, et al (2024)

CRISPR/Cas9-meditated gene knockout in pigs proves that LGALS12 deficiency suppresses the proliferation and differentiation of porcine adipocytes.

Biochimica et biophysica acta. Molecular and cell biology of lipids, 1869(3):159424.

LGALS12, also known as galectin12, belongs to the galectin family with β-galactoside-binding activity. We previously reported that LGALS12 is an important regulator of adipogenesis in porcine adipocytes in vitro, but its value in pig breeding needed to be explored in vivo. In this study, we used CRISPR/Cas9 to construct porcine fetal fibroblasts (PFFs) with a 43 bp deletion in LGALS12 exon 2. Using these PFFs as donor cells, a LGALS12 knockout pig model was generated via somatic cell nuclear transfer. Primary cultures of porcine intramuscular (IM) and subcutaneous (SC) adipocytes were established using cells from LGALS12 knockout pigs and wild-type pigs. A comparison of these cells proved that LGALS12 deficiency suppresses cell proliferation via the RAS-p38MAPK pathway and promotes lipolysis via the PKA pathway in both IM and SC adipocytes. In addition, we observed AKT activation only in IM adipocytes and suppression of the Wnt/β-catenin only in SC adipocytes. Our findings suggest that LGALS12 deficiency affects the adipogenesis of IM and SC adipocytes through different mechanisms.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Sahu S, Sullivan T, Southon E, et al (2023)

Protocol for the saturation and multiplexing of genetic variants using CRISPR-Cas9.

STAR protocols, 4(4):102702.

Here, we present a multiplexed assay for variant effect protocol to assess the functional impact of all possible genetic variations within a particular genomic region. We describe steps for saturation genome editing by designing and cloning of single-guide RNA (sgRNA). We then detail steps for nucleofection of sgRNA, testing drug response on variants, and amplification of genomic DNA for next-generation sequencing. For complete details on the use and execution of this protocol, please refer to Sahu et al.[1].

RevDate: 2024-03-11
CmpDate: 2024-03-11

Ravi NS, George A, KM Mohankumar (2023)

Protocol for arrayed gRNA screening by base editors in mammalian cell lines using lentiviral system.

STAR protocols, 4(4):102668.

Base editing, a CRISPR-based genome engineering technique, enables precise single-nucleotide modifications while minimizing double-strand breaks. Here, we present a protocol for arrayed mutagenesis using base editors to identify regulatory elements within the gamma-globin locus. We describe steps for guide RNA (gRNA) cloning into lentiviral vectors, establishing stable cell lines with base editor expression, transducing gRNAs, and assessing editing efficiency. This protocol can be applied to diverse genomic regions and cell lines for arrayed screening, facilitating genetic research, and target discovery. For complete details on the use and execution of this protocol, please refer to Ravi et al. (2022)[1].

RevDate: 2024-03-11
CmpDate: 2024-03-11

Li Z, Reint G, EM Haapaniemi (2023)

Protocol for editing fibroblasts with in vitro transcribed Cas9 mRNA and profile off-target editing by optimized GUIDE-seq.

STAR protocols, 4(4):102662.

CRISPR-Cas9 gene editing is an efficient technique to modify specific sites/regions of DNA. Delivery of the Cas9 by mRNA is particularly promising in pre-clinical genome editing applications for its transient, nonintegrating feature. However, the off-target of Cas9-gRNA still remains a concern and needs a specific monitor. Here, we present a revised protocol to edit fibroblasts by in vitro transcribed Cas9 mRNA and profile its off-target effect by the optimized GUIDE-seq method. This protocol can also be applied to other cell lines. For complete details on the use and execution of this protocol, please refer to Ganna Reint et al. (2021).[1].

RevDate: 2024-03-11
CmpDate: 2024-03-11

Bril O, Schwarzmueller LJ, Moreno LF, et al (2023)

Identifying essential long non-coding RNAs in cancer using CRISPRi-based dropout screens.

STAR protocols, 4(4):102588.

Long non-coding RNAs (lncRNAs) are emerging as key regulators in the initiation, growth, and progression of cancer. High-throughput CRISPR-based techniques systematically assess the function of genes or regulatory elements present in the human genome. Here, we present a protocol for identifying essential lncRNAs in cancer using CRISPRi-based dropout screens. We describe steps to select target sites, design guide RNAs, and generate CRISPRi cell lines. We then detail the execution and analysis of CRISPRi-based dropout screens.

RevDate: 2024-03-11
CmpDate: 2024-03-11

Wu Y, Sidharta M, Zhong A, et al (2023)

Protocol for the design, conduct, and evaluation of prime editing in human pluripotent stem cells.

STAR protocols, 4(4):102583.

Prime editing introduces single-nucleotide polymorphism changes, small deletions, or insertions at a specific genome site without double-stranded DNA breaks or the need for the donor template. Here, we present a protocol to design, conduct, and evaluate prime editing in human pluripotent stem cells. We describe steps for pegRNA and nicking sgRNA design and cloning, the prime editing tool electroporation, and the efficiency evaluation using Miseq. We elaborate the process of GBA (N370S) mutation induction and correction as an example. For complete details on the use and execution of this protocol, please refer to Li et al. (2022).[1].

RevDate: 2024-03-11
CmpDate: 2024-03-11

Ten Hacken E, Gruber M, Hernández-Sánchez M, et al (2023)

Generation of mouse models carrying B cell restricted single or multiplexed loss-of-function mutations through CRISPR-Cas9 gene editing.

STAR protocols, 4(4):102165.

Here, we present a protocol to generate B cell restricted mouse models of loss-of-function genetic drivers typical of lymphoproliferative disorders, using stem cell engineering of murine strains carrying B cell restricted Cas9 expression. We describe steps for preparing lentivirus expressing sgRNA-mCherry, isolating hematopoietic stem and progenitor cells, and in vitro transduction. We then detail the transplantation of engineered cells into recipient mice and verification of gene edits. These mouse models represent versatile platforms to model complex disease traits typical of lymphoproliferative disorders. For complete details on the use and execution of this protocol, please refer to ten Hacken et al.,[1] ten Hacken et al.,[2] and ten Hacken et al.[3].

RevDate: 2024-03-06

Jung WJ, Park SJ, Cha S, et al (2024)

Factors affecting the cleavage efficiency of the CRISPR-Cas9 system.

Animal cells and systems, 28(1):75-83.

The CRISPR-Cas system stands out as a promising genome editing tool due to its cost-effectiveness and time efficiency compared to other methods. This system has tremendous potential for treating various diseases, including genetic disorders and cancer, and promotes therapeutic research for a wide range of genetic diseases. Additionally, the CRISPR-Cas system simplifies the generation of animal models, offering a more accessible alternative to traditional methods. The CRISPR-Cas9 system can be used to cleave target DNA strands that need to be corrected, causing double-strand breaks (DSBs). DNA with DSBs can then be recovered by the DNA repair pathway that the CRISPR-Cas9 system uses to edit target gene sequences. High cleavage efficiency of the CRISPR-Cas9 system is thus imperative for effective gene editing. Herein, we explore several factors affecting the cleavage efficiency of the CRISPR-Cas9 system. These factors include the GC content of the protospacer-adjacent motif (PAM) proximal and distal regions, single-guide RNA (sgRNA) properties, and chromatin state. These considerations contribute to the efficiency of genome editing.

RevDate: 2024-03-05

Lee Y, Hwang S, Kim W, et al (2024)

CRISPR-aided genome engineering for secondary metabolite biosynthesis in Streptomyces.

Journal of industrial microbiology & biotechnology pii:7619141 [Epub ahead of print].

The demand for discovering novel microbial secondary metabolites is growing to address the limitations in bioactivities such as antibacterial, antifungal, anticancer, anthelmintic, and immunosuppressive functions. Among microbes, the genus Streptomyces holds particular significance for secondary metabolite discovery. Each Streptomyces species typically encodes approximately 30 secondary metabolite biosynthetic gene clusters within its genome, which are mostly uncharacterized in terms of their products and bioactivities. The development of next-generation sequencing has enabled the identification of a large number of potent secondary metabolite biosynthetic gene clusters for novel secondary metabolites that are imbalanced in number compared with discovered secondary metabolites. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has revolutionized the translation of enormous genomic potential into the discovery of secondary metabolites as the most efficient genetic engineering tool for Streptomyces. In this review, the current status of CRISPR/Cas applications in Streptomyces is summarized, with particular focus on the identification of secondary metabolite biosynthesis gene clusters and their potential applications.

RevDate: 2024-03-06
CmpDate: 2024-03-06

Tsuchida CA, Wasko KM, Hamilton JR, et al (2024)

Targeted nonviral delivery of genome editors in vivo.

Proceedings of the National Academy of Sciences of the United States of America, 121(11):e2307796121.

Cell-type-specific in vivo delivery of genome editing molecules is the next breakthrough that will drive biological discovery and transform the field of cell and gene therapy. Here, we discuss recent advances in the delivery of CRISPR-Cas genome editors either as preassembled ribonucleoproteins or encoded in mRNA. Both strategies avoid pitfalls of viral vector-mediated delivery and offer advantages including transient editor lifetime and potentially streamlined manufacturing capability that are already proving valuable for clinical use. We review current applications and future opportunities of these emerging delivery approaches that could make genome editing more efficacious and accessible in the future.

RevDate: 2024-03-07
CmpDate: 2024-03-06

Wang T, Zeng H, Liu Q, et al (2024)

Establishment of RPA-Cas12a-Based Fluorescence Assay for Rapid Detection of Feline Parvovirus.

Polish journal of microbiology, 73(1):39-48.

Feline parvovirus (FPV) is highly infectious for cats and other Felidae and often causes severe damage to young kittens. In this study, we incorporated recombinase polymerase amplification (RPA) and Cas12a-mediated detection and developed an RPA-Cas12a-based real-time or end-point fluorescence detection method to identify the NS1 gene of FPV. The total time of RPA-Cas12a-based fluorescence assay is approximately 25 min. The assay presented a limit of detection (LOD) of 1 copies/μl (25 copies/per reaction), with no cross-reactivity with several feline pathogens. The clinical performance of the assay was examined using total genomic DNA purified from 60 clinical specimens and then compared to results obtained with qPCR detection of FPV with 93.3% positive predictive agreement and 100% negative predictive agreement. Together, the rapid reaction, cost-effectiveness, and high sensitivity make the RPA-Cas12a-based fluorescence assay a fascinating diagnostic tool that will help minimize infection spread through instant detection of FPV.

RevDate: 2024-03-07
CmpDate: 2024-03-06

Hu X, He P, Jiang T, et al (2024)

Development and Evaluation of a Rapid GII Norovirus Detection Method Based on CRISPR-Cas12a.

Polish journal of microbiology, 73(1):89-97.

Norovirus is highly infectious and rapidly transmissible and represents a major pathogen of sporadic cases and outbreaks of acute gastroenteritis worldwide, causing a substantial disease burden. Recent years have witnessed a dramatic increase in norovirus outbreaks in China, significantly higher than in previous years, among which GII norovirus is the predominant prevalent strain. Therefore, rapid norovirus diagnosis is critical for clinical treatment and transmission control. Hence, we developed a molecular assay based on RPA combined with the CRISPER-CAS12a technique targeting the conserved region of the GII norovirus genome, the results of which could be displayed by fluorescence curves and immunochromatographic lateral-flow test strips. The reaction only required approximately 50 min, and the results were visible by the naked eye with a sensitivity reaching 10[2] copies/μl. Also, our method does not cross-react with other common pathogens that cause intestinal diarrhea. Furthermore, this assay was easy to perform and inexpensive, which could be widely applied for detecting norovirus in settings including medical institutions at all levels, particularly township health centers in low-resource areas.

RevDate: 2024-03-06
CmpDate: 2024-03-05

Terrones M, Deben C, Rodrigues-Fortes F, et al (2024)

CRISPR/Cas9-edited ROS1 + non-small cell lung cancer cell lines highlight differential drug sensitivity in 2D vs 3D cultures while reflecting established resistance profiles.

Journal of translational medicine, 22(1):234.

INTRODUCTION: The study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting.

METHODS: In the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR_fit) dose-response curves was the variable used to quantify the cellular response towards TKIs.

RESULTS: Spheroids derived from ROS1[G2032R] cells were significantly more resistant to repotrectinib (AOC fold change = - 7.33), lorlatinib (AOC fold change = - 6.17), ceritinib (AOC fold change = - 2.8) and entrectinib (AOC fold change = - 2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change = - 2.35), entrectinib (AOC fold change = - 2.44) and ceritinib (AOC fold change = - 2.12) in targeting the ROS1 G2032R mutation. ROS1[L2026M] cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change = - 2.19) and entrectinib (spheroid AOC fold change = - 1.98). ROS1[S1986Y] cells were resistant only towards crizotinib in 2D (AOC fold change = - 1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose-response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells.

CONCLUSION: In this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.

RevDate: 2024-03-05
CmpDate: 2024-03-05

Galun E (2024)

[GENE THERAPY: FROM TECHNOLOGY TO REALITY].

Harefuah, 163(2):97-101.

Gene therapy has made major achievements in the last few decades. These were in numerous medical disciplines, including metabolic, oncologic, infectious and regenerative. As of today, regulatory agencies, both in the USA and Europe, approved for clinical usage numerous gene therapy treatments. However, we are still facing a number of significant obstacles including: 1. Efficient delivery systems, 2. Immunological responses, and 3. recently we have learned that many gene therapy approaches are very expensive. The COVID-19 pandemic was a period in which genetic vaccination had proved its efficacy, by which RNA in a synthetic delivery system was very effective. This review will focus on three fast developing technologies in gene therapy: 1. CAR-T cells, 2. CRISPR-Cas and 3. RNAi.

RevDate: 2024-03-08
CmpDate: 2024-03-04

Pacesa M, Pelea O, M Jinek (2024)

Past, present, and future of CRISPR genome editing technologies.

Cell, 187(5):1076-1100.

Genome editing has been a transformative force in the life sciences and human medicine, offering unprecedented opportunities to dissect complex biological processes and treat the underlying causes of many genetic diseases. CRISPR-based technologies, with their remarkable efficiency and easy programmability, stand at the forefront of this revolution. In this Review, we discuss the current state of CRISPR gene editing technologies in both research and therapy, highlighting limitations that constrain them and the technological innovations that have been developed in recent years to address them. Additionally, we examine and summarize the current landscape of gene editing applications in the context of human health and therapeutics. Finally, we outline potential future developments that could shape gene editing technologies and their applications in the coming years.

RevDate: 2024-03-07
CmpDate: 2024-03-04

Chariou PL, Minnar CM, Tandon M, et al (2024)

Generation of murine tumor models refractory to αPD-1/-L1 therapies due to defects in antigen processing/presentation or IFNγ signaling using CRISPR/Cas9.

PloS one, 19(3):e0287733.

Immune checkpoint blockade (ICB) targeting the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1) fails to provide clinical benefit for most cancer patients due to primary or acquired resistance. Drivers of ICB resistance include tumor antigen processing/presentation machinery (APM) and IFNγ signaling mutations. Thus, there is an unmet clinical need to develop alternative therapies for these patients. To this end, we have developed a CRISPR/Cas9 approach to generate murine tumor models refractory to PD-1/-L1 inhibition due to APM/IFNγ signaling mutations. Guide RNAs were employed to delete B2m, Jak1, or Psmb9 genes in ICB-responsive EMT6 murine tumor cells. B2m was deleted in ICB-responsive MC38 murine colon cancer cells. We report a detailed development and validation workflow including whole exome and Sanger sequencing, western blotting, and flow cytometry to assess target gene deletion. Tumor response to ICB and immune effects of gene deletion were assessed in syngeneic mice. This workflow can help accelerate the discovery and development of alternative therapies and a deeper understanding of the immune consequences of tumor mutations, with potential clinical implications.

RevDate: 2024-03-01

Workman RE, Stoltzfus MJ, Keith NC, et al (2024)

Anti-CRISPR proteins trigger a burst of CRISPR-Cas9 expression that enhances phage defense.

Cell reports, 43(3):113849 pii:S2211-1247(24)00177-3 [Epub ahead of print].

CRISPR-Cas immune systems provide bacteria with adaptive immunity against bacteriophages, but they are often transcriptionally repressed to mitigate auto-immunity. In some cases, CRISPR-Cas expression increases in response to a phage infection, but the mechanisms of induction are largely unknown, and it is unclear whether induction occurs strongly and quickly enough to benefit the bacterial host. In S. pyogenes, Cas9 is both an immune effector and auto-repressor of CRISPR-Cas expression. Here, we show that phage-encoded anti-CRISPR proteins relieve Cas9 auto-repression and trigger a rapid increase in CRISPR-Cas levels during a single phage infective cycle. As a result, fewer cells succumb to lysis, leading to a striking survival benefit after multiple rounds of infection. CRISPR-Cas induction also reduces lysogeny, thereby limiting a route for horizontal gene transfer. Altogether, we show that Cas9 is not only a CRISPR-Cas effector and repressor but also a phage sensor that can mount an anti-anti-CRISPR transcriptional response.

RevDate: 2024-03-08
CmpDate: 2024-03-08

Merenciano M, Aguilera L, J González (2023)

Two-step CRISPR-Cas9 protocol for transposable element deletion in D. melanogaster natural populations.

STAR protocols, 4(3):102501.

We present a protocol for generating a precise deletion, without altering the genetic background of the strain, of a transposable element (TE) in a natural population of Drosophila melanogaster using two steps of CRISPR-Cas9 homology-directed repair. We describe steps for replacing the TE by a fluorescent marker and for subsequent marker removal using single-guide RNAs, repair plasmids, and microinjection. We also detail steps for screening the deletion of the TE and generating a homozygous mutant strain. For complete details on the use and execution of this protocol, please refer to Merenciano and Gonzalez.[1].

RevDate: 2024-03-08
CmpDate: 2024-03-08

Zhang Z (2023)

Generation of epitope tag knock-in mice with CRISPR-Cas9 to study the function of endogenous proteins.

STAR protocols, 4(3):102518.

Studying endogenous proteins in mice has provided numerous insights into the physiological and pathological roles of these proteins. However, the availability and specificity of protein-specific antibodies often limit such studies. Here we present a protocol for generating epitope tag knock-in mice with CRISPR-Cas9-mediated gene editing. We discuss key considerations for tag selection and knock-in location and provide insights into CRISPR design. Subsequently, we outline the sequential steps involved in knock-in mouse generation, genotyping, and validation and explore potential applications. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).[1].

RevDate: 2024-03-08
CmpDate: 2024-03-08

Ludwik KA, Telugu N, Schommer S, et al (2023)

ASSURED-optimized CRISPR protocol for knockout/SNP knockin in hiPSCs.

STAR protocols, 4(3):102406.

CRISPR-Cas9 technology coupled with human induced pluripotent stem cells allows precise disease modeling in pluripotent cells and subsequently derived specialized cell types. Here, we present an optimized CRISPR-Cas9 pipeline, ASSURED (affordable, successful, specific, user-friendly, rapid, efficient, and deliverable), to produce gene-modified single-cell-derived knockout or single-nucleotide-polymorphism-modified knockin hiPSCs clones. We describe steps for analyzing targeted genomic sequence and designing guide RNAs and homology repair template. We then detail the CRISPR-Cas9 delivery workflow, evaluation of editing efficiency, and automated cell isolation followed by clone screening.

RevDate: 2024-03-08
CmpDate: 2024-03-08

Wu L, Tan JC, NRJ Gascoigne (2023)

Generation of gene-of-interest double allele knockout clones in primary human T cells by CRISPR.

STAR protocols, 4(3):102445.

Gene-of-interest (GOI) knockout is an important technique to study the genetic mechanisms of T cells. Here, we present a protocol to generate GOI double allele gene knockouts in primary human T cells by CRISPR, thus depleting proteins of interest expressed intracellularly or extracellularly in primary T cells. We describe steps for gRNA selection and efficiency validation, homology-directed repair (HDR) DNA template design and cloning, and genome editing and HDR gene insertion. We then detail clone isolation and GOI knockout validation. For complete details on the use and execution of this protocol, please refer to Wu et al.[1].

RevDate: 2024-03-08
CmpDate: 2024-03-08

du Halgouet A, Darbois A, Alphonse A, et al (2023)

Protocol to expand and CRISPR-Cas9 genomic edit murine MAIT cells for subsequent in vivo studies.

STAR protocols, 4(3):102419.

Generating knockout mice for target molecules in specific T cell populations, without subset-specific promoters, is time-consuming and costly. Here, we describe steps for enriching mucosal-associated invariant T cells from the thymus, expanding them in vitro and performing a CRISPR-Cas9 knockout. We then detail procedure for injecting the knockout cells into wounded Cd3ε[-/-] mice and characterizing them in the skin. For complete details on the use and execution of this protocol, please refer to du Halgouet et al. (2023).[1].

RevDate: 2024-03-08
CmpDate: 2024-03-08

Zeng X, Wang S, Liang M, et al (2023)

An in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments.

STAR protocols, 4(3):102435.

Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria). Here, we present an in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments. We describe steps for crRNA design and preparation, genomic DNA isolation, and CRISPR-Cas12a cleavage and capture plasmid construction and linearization. We then detail target BGC and plasmid DNA ligation and transformation and screening for positive clones. For complete details on the use and execution of this protocol, please refer to Liang et al.[1].

RevDate: 2024-03-08
CmpDate: 2024-03-08

Geyer F, Geyer M, Klapproth S, et al (2023)

Protocol for generating monoclonal CRISPR-Cas9-mediated knockout cell lines using RNPs and lipofection in HNSCC cells.

STAR protocols, 4(3):102366.

CRISPR-Cas9 is a powerful technology for accurate and optimizable genome editing. Here, we present a protocol for generating monoclonal knockout (KO) cell lines using CRISPR-Cas9, ribonucleoprotein complexes (RNPs), and lipofection in adherent HNSCC cells from start to finish. We describe steps for choosing the suitable guide and primer design, preparation of guide-RNA (gRNA), lipofection of RNP complexes in HN cells, and single-cell cloning with limiting dilution. We then detail PCR and DNA purification and the selection and verification of monoclonal KO cell lines.

RevDate: 2024-03-08
CmpDate: 2024-03-08

Shibata Y, Okumura A, Mochii M, et al (2023)

Protocols for transgenesis at a safe harbor site in the Xenopus laevis genome using CRISPR-Cas9.

STAR protocols, 4(3):102382.

We have established a new transgenesis protocol based on CRISPR-Cas9, "New and Easy XenopusTransgenesis (NEXTrans)," and identified a novel safe harbor site in African clawed frogs, Xenopus laevis. We describe steps in detail for the construction of NEXTrans plasmid and guide RNA, CRISPR-Cas9-mediated NEXTrans plasmid integration into the locus, and its validation by genomic PCR. This improved strategy allows us to simply generate transgenic animals that stably express the transgene. For complete details on the use and execution of this protocol, please refer to Shibata et al. (2022).[1].

RevDate: 2024-03-04
CmpDate: 2024-03-04

Song MK, YS Kim (2024)

Targeted Modification of Epigenetic Marks Using CRISPR/dCas9-SunTag-Based Modular Epigenetic Toolkit.

Methods in molecular biology (Clifton, N.J.), 2761:81-91.

The epigenome, consisting of chemical modifications to DNA and histone proteins, can alter gene expression. Clustered regularly interspaced short palindromic repeats/dead CRISPR-associated protein 9 (CRISPR/dCas9) systems enable precise target gene-specific gene modulation by attaching different "effector" domains to the dCas9 protein to activate or repress specific genes. CRISPR/dCas9-SunTag is an improved system version, allowing more efficient and precise gene activation or repression by recruiting multiple copies of the protein of interest. A CRISPR/dCas9-SunTag-based modular epigenetic toolkit was developed, enabling gene-specific epigenetic architecture modulation. This protocol generated a stable SH-SY5Y cell line expressing the CRISPR/dCas9-SunTag-JARID1A system to study H3K4Me3-mediated promoter regulation at a 200-400 bp of fine resolution. The procedure involved designing sgRNAs, subcloning dCas9-5XGCN4 into pLvx-DsRed, validating epigenetic mark changes with ChIP, and validating gene expression changes with RT-qPCR. This epigenetic toolkit is valuable for researchers to understand the relationship between gene-specific epigenetic modifications and gene expression.

RevDate: 2024-03-01

Hua HM, Xu JF, Huang XS, et al (2024)

Low-Toxicity and High-Efficiency Streptomyces Genome Editing Tool Based on the Miniature Type V-F CRISPR/Cas Nuclease AsCas12f1.

Journal of agricultural and food chemistry [Epub ahead of print].

Genome editing tools based on SpCas9 and FnCpf1 have facilitated strain improvements for natural product production and novel drug discovery in Streptomyces. However, due to high toxicity, their editing requires high DNA transformation efficiency, which is unavailable in most streptomycetes. The transformation efficiency of an all-in-one editing tool based on miniature Cas nuclease AsCas12f1 was significantly higher than those of SpCas9 and FnCpf1 in tested streptomycetes, which is due to its small size and weak DNA cleavage activity. Using this tool, in Streptomyces coelicolor, we achieved 100% efficiency for single gene or gene cluster deletion and 46.7 and 40% efficiency for simultaneous deletion of two genes and two gene clusters, respectively. AsCas12f1 was successfully extended to Streptomyces hygroscopicus SIPI-054 for efficient genome editing, in which SpCas9/FnCpf1 does not work well. Collectively, this work offers a low-toxicity, high-efficiency genome editing tool for streptomycetes, particularly those with low DNA transformation efficiency.

RevDate: 2024-03-04
CmpDate: 2024-03-04

Yang Z, Zhang Z, Li J, et al (2024)

CRISPRlnc: a machine learning method for lncRNA-specific single-guide RNA design of CRISPR/Cas9 system.

Briefings in bioinformatics, 25(2):.

CRISPR/Cas9 is a promising RNA-guided genome editing technology, which consists of a Cas9 nuclease and a single-guide RNA (sgRNA). So far, a number of sgRNA prediction softwares have been developed. However, they were usually designed for protein-coding genes without considering that long non-coding RNA (lncRNA) genes may have different characteristics. In this study, we first evaluated the performances of a series of known sgRNA-designing tools in the context of both coding and non-coding datasets. Meanwhile, we analyzed the underpinnings of their varied performances on the sgRNA's specificity for lncRNA including nucleic acid sequence, genome location and editing mechanism preference. Furthermore, we introduce a support vector machine-based machine learning algorithm named CRISPRlnc, which aims to model both CRISPR knock-out (CRISPRko) and CRISPR inhibition (CRISPRi) mechanisms to predict the on-target activity of targets. CRISPRlnc combined the paired-sgRNA design and off-target analysis to achieve one-stop design of CRISPR/Cas9 sgRNAs for non-coding genes. Performance comparison on multiple datasets showed that CRISPRlnc was far superior to existing methods for both CRISPRko and CRISPRi mechanisms during the lncRNA-specific sgRNA design. To maximize the availability of CRISPRlnc, we developed a web server (http://predict.crisprlnc.cc) and made it available for download on GitHub.

RevDate: 2024-03-07
CmpDate: 2024-03-04

Parums DV (2024)

Editorial: First Regulatory Approvals for CRISPR-Cas9 Therapeutic Gene Editing for Sickle Cell Disease and Transfusion-Dependent β-Thalassemia.

Medical science monitor : international medical journal of experimental and clinical research, 30:e944204.

In 2020, Emmanuelle Charpentier and Jennifer Doudna were awarded the Nobel Prize in Chemistry for their research on the endonuclease, clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (CRISPR-Cas9) method for DNA editing. On 16 November 2023, the UK Medicines and Healthcare Products Regulatory Agency (MHRA) was the first to approve the CRISPR-Cas9 gene editing therapy, Casgevy (exagamglogene autotemcel), for the treatment of patients with transfusion-dependent b-thalassemia and the treatment of sickle cell disease in patients aged ≥12 years with recurrent vaso-occlusive crises. On 8 December 2023, the US Food and Drug Administration (FDA) approved both Casgevy and Lyfgenia (lovotibeglogene autotemcel) for patients with sickle cell disease. On 15 December 2023, the European Medicines Agency (EMA) approved Casgevy for sickle cell disease and transfusion-dependent ß-thalassemia. This Editorial aims to present an update on the landmark first regulatory approvals of CRISPR-Cas9 for patients with sickle cell disease and transfusion-dependent b-thalassemia and the potential challenges for therapeutic gene (DNA) editing.

RevDate: 2024-03-07
CmpDate: 2024-03-07

Bai M, Lin W, Peng C, et al (2024)

Expressing a human RNA demethylase as an assister improves gene-editing efficiency in plants.

Molecular plant, 17(3):363-366.

RevDate: 2024-03-04
CmpDate: 2024-03-04

Caforio M, Iacovelli S, Quintarelli C, et al (2024)

GMP-manufactured CRISPR/Cas9 technology as an advantageous tool to support cancer immunotherapy.

Journal of experimental & clinical cancer research : CR, 43(1):66.

BACKGROUND: CRISPR/Cas9 system to treat human-related diseases has achieved significant results and, even if its potential application in cancer research is improving, the application of this approach in clinical practice is still a nascent technology.

MAIN BODY: CRISPR/Cas9 technology is not yet used as a single therapy to treat tumors but it can be combined with traditional treatment strategies to provide personalized gene therapy for patients. The combination with chemotherapy, radiation and immunotherapy has been proven to be a powerful means of screening, identifying, validating and correcting tumor targets. Recently, CRISPR/Cas9 technology and CAR T-cell therapies have been integrated to open novel opportunities for the production of more efficient CAR T-cells for all patients. GMP-compatible equipment and reagents are already available for several clinical-grade systems at present, creating the basis and framework for the accelerated development of novel treatment methods.

CONCLUSION: Here we will provide a comprehensive collection of the actual GMP-grade CRISPR/Cas9-mediated approaches used to support cancer therapy highlighting how this technology is opening new opportunities for treating tumors.

RevDate: 2024-03-04
CmpDate: 2024-03-04

Lee SR, Lee KL, Song SH, et al (2024)

Generation of Fel d 1 chain 2 genome-edited cats by CRISPR-Cas9 system.

Scientific reports, 14(1):4987.

Allergens from domestic cats (Felis catus) cause allergy-related health problems worldwide. Fel d 1 is a major allergen that causes severe allergic reactions in humans, including rhinitis, conjunctivitis, and life-threatening asthma. Therefore, patients with cat allergies anticipate hypoallergenic cats. We successfully generated Fel d 1 chain 2 (CH2) genome-edited cats using the CRISPR-Cas9 system in this study. T7 endonuclease 1 assay and Sanger sequencing were used to confirm the mutation in CH2 genome-edited cats. Fel d 1 level in CH2 genome-edited cats were assessed by enzyme-linked immunosorbent assay (ELISA). Remarkably, ELISA showed that the level of Fel d 1 in the CH2 homozygous genome-edited cat (Name: Alsik) was extremely low compared with that in wild type domestic cats and could be hypoallergenic cats. Additionally, we successfully cloned the CH2 homozygous genome-edited cat using cytoplasm injection clone technology. The cloned CH2 homozygous genome-edited cat was verified using microsatellite analysis. Creating hypoallergenic cats using the CRISPR-Cas9 system is a significant step forward because these cats can safely approach allergic patients.

RevDate: 2024-03-04
CmpDate: 2024-03-04

Song J, Zhuang Y, C Yi (2024)

Programmable RNA base editing via targeted modifications.

Nature chemical biology, 20(3):277-290.

Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editors are powerful tools in biology and hold great promise for the treatment of human diseases. Advanced DNA base editing tools, such as cytosine base editor and adenine base editor, have been developed to correct permanent mistakes in genetic material. However, undesired off-target edits would also be permanent, which poses a considerable risk for therapeutics. Alternatively, base editing at the RNA level is capable of correcting disease-causing mutations but does not lead to lasting genotoxic effects. RNA base editors offer temporary and reversible therapies and have been catching on in recent years. Here, we summarize some emerging RNA editors based on A-to-inosine, C-to-U and U-to-pseudouridine changes. We review the programmable RNA-targeting systems as well as modification enzyme-based effector proteins and highlight recent technological breakthroughs. Finally, we compare editing tools, discuss limitations and opportunities, and provide insights for the future directions of RNA base editing.

RevDate: 2024-03-01
CmpDate: 2024-03-01

Lin C, Zhou J, Gao N, et al (2024)

Establishing a pulmonary aspergillus fumigatus infection diagnostic platform based on RPA-CRISPR-Cas12a.

World journal of microbiology & biotechnology, 40(4):116.

In this study, we devised a diagnostic platform harnessing a combination of recombinase polymerase amplification (RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system. Notably, this platform obviates the need for intricate equipment and finds utility in diverse settings. Two result display methods were incorporated in this investigation: the RPA-Cas12a-fluorescence method and the RPA-Cas12a-LFS (lateral flow strip). Upon validation, both display platforms exhibited no instances of cross-reactivity, with seven additional types of fungal pathogens responsible for respiratory infections. The established detection limit was ascertained to be as low as 10[2] copies/µL. In comparison to fluorescence quantitative PCR, the platform demonstrated a sensitivity of 96.7%, a specificity of 100%, and a consistency rate of 98.0%.This platform provides expeditious, precise, and on-site detection capabilities, thereby rendering it a pivotal diagnostic instrument amenable for deployment in primary healthcare facilities and point-of-care settings.

RevDate: 2024-03-02
CmpDate: 2024-03-01

Deng X, Sun W, Li X, et al (2024)

An anti-CRISPR that represses its own transcription while blocking Cas9-target DNA binding.

Nature communications, 15(1):1806.

AcrIIA15 is an anti-CRISPR (Acr) protein that inhibits Staphylococcus aureus Cas9 (SaCas9). Although previous studies suggested it has dual functions, the structural and biochemical basis for its two activities remains unclear. Here, we determined the cryo-EM structure of AcrIIA15 in complex with SaCas9-sgRNA to reveal the inhibitory mechanism of the Acr's C-terminal domain (CTD) in mimicking dsDNA to block protospacer adjacent motif (PAM) recognition. For the N-terminal domain (NTD), our crystal structures of the AcrIIA15-promoter DNA show that AcrIIA15 dimerizes through its NTD to recognize double-stranded (ds) DNA. Further, AcrIIA15 can simultaneously bind to both SaCas9-sgRNA and promoter DNA, creating a supercomplex of two Cas9s bound to two CTDs converging on a dimer of the NTD bound to a dsDNA. These findings shed light on AcrIIA15's inhibitory mechanisms and its autoregulation of transcription, enhancing our understanding of phage-host interactions and CRISPR defense.

RevDate: 2024-03-02

Yu J, Tang M, Zhou Z, et al (2024)

Biologically produced and metal-organic framework delivered dual-cut CRISPR/Cas9 system for efficient gene editing and sensitized cancer therapy.

Acta biomaterialia pii:S1742-7061(24)00101-6 [Epub ahead of print].

Manipulation of the lactate metabolism is an efficient way for cancer treatment given its involvement in cancer development, metastasis, and immune escape. However, most of the inhibitors of lactate transport carriers suffer from poor specificity. Herein, we use the CRISPR/Cas9 system to precisely downregulate the monocarboxylate carrier 1 (MCT1) expression. To avoid the self-repairing during the gene editing process, a dual-Cas9 ribonucleoproteins (duRNPs) system is generated using the biological fermentation method and delivered into cells by the zeolitic imidazolate framework-8 (ZIF-8) nanoparticles, enabling precise removal of a specific DNA fragment from the genome. For efficient cancer therapy, a specific glucose transporter 1 inhibitor (BAY-876) is co-delivered with the duRNPs, forming BAY/duRNPs@ZIF-8 nanoparticle. ZIF-8 nanoparticles can deliver the duRNPs into cells within 1 h, which efficiently downregulates the MCT1 expression, and prohibits lactate influx. Through simultaneous inhibition of the lactate and glucose influx, BAY/duRNPs@ZIF-8 prohibits ATP generation, arrests cell cycle, inhibits cell proliferation, and finally induces cellular apoptosis both in vitro and in vivo. Consequently, we demonstrate that the biologically produced duRNPs delivered into cells by the nonviral ZIF-8 carrier have expanded the CRISPR/Cas gene editing toolbox and elevated the gene editing efficiency, which will promote biological studies and clinical applications. STATEMENT OF SIGNIFICANCE: The CRISPR/Cas9 system, widely used as an efficient gene editing tool, faces a challenge due to cells' ability to self-repair. To address this issue, a strategy involving dual-cutting of the genome DNA has been designed and implemented. This strategy utilizes biologically produced dual-ribonucleoproteins delivered by a metal-organic framework. The effectiveness of this dual-cut CRISPR-Cas9 system has been demonstrated through a therapeutic approach targeting the simultaneous inhibition of lactate and glucose influx in cancer cells. The utilization of the dual-cut gene editing strategy has provided valuable insights into gene editing and expanded the toolbox of the CRISPR/Cas-based gene editing system. It has the potential to enable more efficient and precise manipulation of specific protein expression in the future.

RevDate: 2024-03-06
CmpDate: 2024-03-06

Cao M, Bian X, Ji Z, et al (2024)

Heparin Specifically Inhibits CRISPR/Cas12 Activation, Enabling Ultrasensitive Heparin Detection and Gene Editing Regulation.

Analytical chemistry, 96(9):3970-3978.

Heparin is a highly sulfated linear glycosaminoglycan that is used as an anticoagulant to prevent and treat thrombotic diseases. Herein, we find that heparin specifically inhibits the activation of the Cas12 protein through the competitive binding of heparin and crRNA to Cas12. Studies illustrate that heparin's high molecular weight and strong negative charge are critical parameters for its inhibitory effect. This unexpected finding was engineered for the detection of heparin, affording a low detection limit of 0.36 ng/mL for fluorometric quantification. We further developed a rapid lateral flow-based system named HepaStrip (heparin strip), allowing heparin monitoring in clinical samples within 20 min. Finally, in vivo investigations revealed that heparin can regulate gene editing with the clusters of the regularly spaced short palindromic repeat (CRISPR)/Cas12 system in Escherichia coli. Heparin blocks the formation of Cas12-crRNA ribonucleoprotein, allowing the application of CRISPR for rapid and field-deployable detection of heparin and unleashing the potential use of heparin in future anti-CRISPR applications.

RevDate: 2024-03-01
CmpDate: 2024-02-29

Gupta P, Goswami SG, Kumari G, et al (2024)

Development of pathophysiologically relevant models of sickle cell disease and β-thalassemia for therapeutic studies.

Nature communications, 15(1):1794.

Ex vivo cellular system that accurately replicates sickle cell disease and β-thalassemia characteristics is a highly sought-after goal in the field of erythroid biology. In this study, we present the generation of erythroid progenitor lines with sickle cell disease and β-thalassemia mutation using CRISPR/Cas9. The disease cellular models exhibit similar differentiation profiles, globin expression and proteome dynamics as patient-derived hematopoietic stem/progenitor cells. Additionally, these cellular models recapitulate pathological conditions associated with both the diseases. Hydroxyurea and pomalidomide treatment enhanced fetal hemoglobin levels. Notably, we introduce a therapeutic strategy for the above diseases by recapitulating the HPFH3 genotype, which reactivates fetal hemoglobin levels and rescues the disease phenotypes, thus making these lines a valuable platform for studying and developing new therapeutic strategies. Altogether, we demonstrate our disease cellular systems are physiologically relevant and could prove to be indispensable tools for disease modeling, drug screenings and cell and gene therapy-based applications.

RevDate: 2024-03-01

Boan AF, Delgadin TH, Canosa LF, et al (2024)

Loss of function in somatostatin receptor 5 has no impact on the growth of medaka fish due to compensation by the other paralogs.

General and comparative endocrinology, 351:114478 pii:S0016-6480(24)00038-8 [Epub ahead of print].

Somatic growth in vertebrates is regulated endocrinologically by the somatotropic axis, headed by the growth hormone (GH) and the insulin growth factor-I (IGF-I). Somatostatin (Sst), a peptide hormone synthesized in the hypothalamus, modulates GH actions through its receptors (Sstr). Four Sstr subtypes (Sstr 1-3 and 5) have been identified in teleosts. However, little is known about whether they have a specific function or tissue expression. The aim of this study was to determine the role of sstr2 and sstr5 in the growth of the medaka (Oryzias latipes). The assessed expression pattern across diverse tissues highlighted greater prevalence of sstr1 and sstr3 in brain, intestine and muscle than in pituitary or liver. The expression of sstr2 was high in all the tissues tested, while sstr5 was predominantly expressed in the pituitary gland. A CRISPR/Cas9 sstr5 mutant with loss of function (sstr5[-/-]) was produced. Assessment of sstr5[-/-] indicated no significant difference with the wild type regarding growth parameters such as standard length, body depth, or peduncle depth. Furthermore, the functional loss of sstr5 had no impact on the response to a nutritional challenge. The fact that several sstr subtypes were upregulated in different tissues in sstr5[-/-] medaka suggests that in the mutant fish, there may be a compensatory effect on the different tissues, predominantly by sstr1 in the liver, brain and pituitary, with sstr2 being upregulated in pituitary and liver, and sstr3 only presenting differential expression in the brain. Analysis of the sstr subtype and the sstr5[-/-] fish showed that sstr5 was not the only somatostatin receptor responsible for Sst-mediated Gh regulation.

RevDate: 2024-02-27

Teng F, Guo F, Feng J, et al (2024)

Distribution analysis of TRH in Bactrocera dorsalis using a CRISPR/Cas9-mediated reporter knock-in strain.

Insect molecular biology [Epub ahead of print].

Although the study of many genes and their protein products is limited by the availability of high-quality antibodies, this problem could be solved by fusing a tag/reporter to an endogenous gene using a gene-editing approach. The type II bacterial CRISPR/Cas system has been demonstrated to be an efficient gene-targeting technology for many insects, including the oriental fruit fly Bactrocera dorsalis. However, knocking in, an important editing method of the CRISPR/Cas9 system, has lagged in its application in insects. Here, we describe a highly efficient homology-directed genome editing system for B. dorsalis that incorporates coinjection of embryos with Cas9 protein, guide RNA and a short single-stranded oligodeoxynucleotide donor. This one-step procedure generates flies carrying V5 tag (42 bp) in the BdorTRH gene. In insects, as in other invertebrates and in vertebrates, the neuronal tryptophan hydroxylase (TRH) gene encodes the rate-limiting enzyme for serotonin biosynthesis in the central nervous system. Using V5 monoclonal antibody, the distribution of TRH in B. dorsalis at different developmental stages was uncovered. Our results will facilitate the generation of insects carrying precise DNA inserts in endogenous genes and will lay foundation for the investigation of the neural mechanisms underlying the serotonin-mediated behaviour of B. dorsalis.

RevDate: 2024-02-27

Kogay R, Wolf YI, EV Koonin (2024)

Defense systems and horizontal gene transfer in bacteria.

bioRxiv : the preprint server for biology.

Horizontal gene transfer (HGT) is a fundamental process in the evolution of prokaryotes, making major contributions to diversification and adaptation. Typically, HGT is facilitated by mobile genetic elements (MGEs), such as conjugative plasmids and phages that generally impose fitness costs on their hosts. However, a substantial fraction of bacterial genes is involved in defense mechanisms that limit the propagation of MGEs, raising the possibility that they can actively restrict HGT. Here we examine whether defense systems curb HGT by exploring the connections between HGT rate and the presence of 73 defense systems in 12 bacterial species. We found that only 6 defense systems, 3 of which are different CRISPR-Cas subtypes, are associated with the reduced gene gain rate on the scale of species evolution. The hosts of such defense systems tend to have a smaller pangenome size and harbor fewer phage-related genes compared to genomes lacking these systems, suggesting that these defense mechanisms inhibit HGT by limiting the integration of prophages. We hypothesize that restriction of HGT by defense systems is species-specific and depends on various ecological and genetic factors, including the burden of MGEs and fitness effect of HGT in bacterial populations.

RevDate: 2024-02-29
CmpDate: 2024-02-28

Zheng Y, Li Y, Zhou K, et al (2024)

Precise genome-editing in human diseases: mechanisms, strategies and applications.

Signal transduction and targeted therapy, 9(1):47.

Precise genome-editing platforms are versatile tools for generating specific, site-directed DNA insertions, deletions, and substitutions. The continuous enhancement of these tools has led to a revolution in the life sciences, which promises to deliver novel therapies for genetic disease. Precise genome-editing can be traced back to the 1950s with the discovery of DNA's double-helix and, after 70 years of development, has evolved from crude in vitro applications to a wide range of sophisticated capabilities, including in vivo applications. Nonetheless, precise genome-editing faces constraints such as modest efficiency, delivery challenges, and off-target effects. In this review, we explore precise genome-editing, with a focus on introduction of the landmark events in its history, various platforms, delivery systems, and applications. First, we discuss the landmark events in the history of precise genome-editing. Second, we describe the current state of precise genome-editing strategies and explain how these techniques offer unprecedented precision and versatility for modifying the human genome. Third, we introduce the current delivery systems used to deploy precise genome-editing components through DNA, RNA, and RNPs. Finally, we summarize the current applications of precise genome-editing in labeling endogenous genes, screening genetic variants, molecular recording, generating disease models, and gene therapy, including ex vivo therapy and in vivo therapy, and discuss potential future advances.

RevDate: 2024-03-05
CmpDate: 2024-02-28

Chen K, Stahl EC, Kang MH, et al (2024)

Engineering self-deliverable ribonucleoproteins for genome editing in the brain.

Nature communications, 15(1):1727.

The delivery of CRISPR ribonucleoproteins (RNPs) for genome editing in vitro and in vivo has important advantages over other delivery methods, including reduced off-target and immunogenic effects. However, effective delivery of RNPs remains challenging in certain cell types due to low efficiency and cell toxicity. To address these issues, we engineer self-deliverable RNPs that can promote efficient cellular uptake and carry out robust genome editing without the need for helper materials or biomolecules. Screening of cell-penetrating peptides (CPPs) fused to CRISPR-Cas9 protein identifies potent constructs capable of efficient genome editing of neural progenitor cells. Further engineering of these fusion proteins establishes a C-terminal Cas9 fusion with three copies of A22p, a peptide derived from human semaphorin-3a, that exhibits substantially improved editing efficacy compared to other constructs. We find that self-deliverable Cas9 RNPs generate robust genome edits in clinically relevant genes when injected directly into the mouse striatum. Overall, self-deliverable Cas9 proteins provide a facile and effective platform for genome editing in vitro and in vivo.

RevDate: 2024-02-29
CmpDate: 2024-02-28

Wang G, Wang F, Xu Z, et al (2024)

Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture.

Genome biology, 25(1):59.

BACKGROUND: CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly.

RESULTS: Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1 with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1 to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1[L86P] and GhTFL1[K53G+S78G] mutations, we find that the GhTFL1[L86P] mutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function.

CONCLUSIONS: This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.

RevDate: 2024-02-26

Kang W, Xiao F, Zhu X, et al (2024)

Engineering Anti-CRISPR Proteins to Create Cas12a Protein Switches for Activatable Genome Editing and Viral Protease Detection.

Angewandte Chemie (International ed. in English) [Epub ahead of print].

Proteins capable of switching between distinct active states in response to biochemical cues are ideal for sensing and controlling biological processes. Activatable CRISPR-Cas systems are significant in precise genetic manipulation and sensitive molecular diagnostics, yet directly controlling Cas protein function remains challenging. Herein, we explore anti-CRISPR (Acr) proteins as modules to create synthetic Cas protein switches (CasPSs) based on computational chemistry-directed rational protein interface engineering. Guided by molecular fingerprint analysis, electrostatic potential mapping, and binding free energy calculations, we rationally engineer the molecular interaction interface between Cas12a and its cognate Acr proteins (AcrVA4 and AcrVA5) to generate a series of orthogonal protease-responsive CasPSs. These CasPSs enable the conversion of specific proteolytic events into activation of Cas12a function with high switching ratios (up to 34.3-fold). These advancements enable specific proteolysis-inducible genome editing in mammalian cells and sensitive detection of viral protease activities during virus infection. This work provides a promising strategy for developing CRISPR-Cas tools for controllable gene manipulation and regulation and clinical diagnostics.

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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.

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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.

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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.

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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.

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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.

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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.

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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

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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.

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