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Bibliography on: CRISPR-Cas

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ESP: PubMed Auto Bibliography 09 Apr 2020 at 01:37 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®)

RevDate: 2020-04-08

Iranzo J, Faure G, Wolf YI, et al (2020)

Game-Theoretical Modeling of Interviral Conflicts Mediated by Mini-CRISPR Arrays.

Frontiers in microbiology, 11:381.

All cellular organisms coevolve with multiple viruses, so that both virus-host and intervirus conflicts are major factors of evolution. Accordingly, hosts evolve multiple, elaborate defense systems and viruses respond by evolving means of antidefense. Although less thoroughly characterized, several dedicated mechanisms of intervirus competition have been described as well. Recently, the genomes of some bacterial and archaeal viruses have been shown to harbor CRISPR mini-arrays that typically contain a single spacer targeting a closely related virus. The involvement of mini-arrays in an intervirus conflict has been experimentally demonstrated for a pair of archaeal viruses. We model the evolution of virus-encoded CRISPR mini-arrays using a game theoretical approach. Analysis of the model reveals multiple equilibria that include mutual targeting, unidirectional targeting, no targeting, cyclic polymorphism, and bistability. The choice between these evolutionary regimes depends on the model parameters including the coinfection frequency, differential productivity of the conflicting viruses, and the fitness cost of mini-arrays. At high coinfection frequencies, the model becomes a version of the Prisoner's dilemma in which defection, i.e., mutual targeting between the competing viruses, is the winning strategy.

RevDate: 2020-04-08

Menchaca A, Dos Santos-Neto PC, Souza-Neves M, et al (2020)

Otoferlin gene editing in sheep via CRISPR-assisted ssODN-mediated Homology Directed Repair.

Scientific reports, 10(1):5995 pii:10.1038/s41598-020-62879-y.

Different mutations of the OTOF gene, encoding for otoferlin protein expressed in the cochlear inner hair cells, induces a form of deafness that is the major cause of nonsyndromic recessive auditory neuropathy spectrum disorder in humans. We report the generation of the first large animal model of OTOF mutations using the CRISPR system associated with different Cas9 components (mRNA or protein) assisted by single strand oligodeoxynucleotides (ssODN) to induce homology-directed repair (HDR). Zygote microinjection was performed with two sgRNA targeting exon 5 and 6 associated to Cas9 mRNA or protein (RNP) at different concentrations in a mix with an ssODN template targeting HDR in exon 5 containing two STOP sequences. A total of 73 lambs were born, 13 showing indel mutations (17.8%), 8 of which (61.5%) had knock-in mutations by HDR. Higher concentrations of Cas9-RNP induced targeted mutations more effectively, but negatively affected embryo survival and pregnancy rate. This study reports by the first time the generation of OTOF disrupted sheep, which may allow better understanding and development of new therapies for human deafness related to genetic disorders. These results support the use of CRISPR/Cas system assisted by ssODN as an effective tool for gene editing in livestock.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Liu Q, Cheng X, Liu G, et al (2020)

Deep learning improves the ability of sgRNA off-target propensity prediction.

BMC bioinformatics, 21(1):51.

BACKGROUND: CRISPR/Cas9 system, as the third-generation genome editing technology, has been widely applied in target gene repair and gene expression regulation. Selection of appropriate sgRNA can improve the on-target knockout efficacy of CRISPR/Cas9 system with high sensitivity and specificity. However, when CRISPR/Cas9 system is operating, unexpected cleavage may occur at some sites, known as off-target. Presently, a number of prediction methods have been developed to predict the off-target propensity of sgRNA at specific DNA fragments. Most of them use artificial feature extraction operations and machine learning techniques to obtain off-target scores. With the rapid expansion of off-target data and the rapid development of deep learning theory, the existing prediction methods can no longer satisfy the prediction accuracy at the clinical level.

RESULTS: Here, we propose a prediction method named CnnCrispr to predict the off-target propensity of sgRNA at specific DNA fragments. CnnCrispr automatically trains the sequence features of sgRNA-DNA pairs with GloVe model, and embeds the trained word vector matrix into the deep learning model including biLSTM and CNN with five hidden layers. We conducted performance verification on the data set provided by DeepCrispr, and found that the auROC and auPRC in the "leave-one-sgRNA-out" cross validation could reach 0.957 and 0.429 respectively (the Pearson value and spearman value could reach 0.495 and 0.151 respectively under the same settings).

CONCLUSION: Our results show that CnnCrispr has better classification and regression performance than the existing states-of-art models. The code for CnnCrispr can be freely downloaded from https://github.com/LQYoLH/CnnCrispr.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Carpenter MD, Hu Q, Bond AM, et al (2020)

Nr4a1 suppresses cocaine-induced behavior via epigenetic regulation of homeostatic target genes.

Nature communications, 11(1):504.

Endogenous homeostatic mechanisms can restore normal neuronal function following cocaine-induced neuroadaptations. Such mechanisms may be exploited to develop novel therapies for cocaine addiction, but a molecular target has not yet been identified. Here we profiled mouse gene expression during early and late cocaine abstinence to identify putative regulators of neural homeostasis. Cocaine activated the transcription factor, Nr4a1, and its target gene, Cartpt, a key molecule involved in dopamine metabolism. Sustained activation of Cartpt at late abstinence was coupled with depletion of the repressive histone modification, H3K27me3, and enrichment of activating marks, H3K27ac and H3K4me3. Using both CRISPR-mediated and small molecule Nr4a1 activation, we demonstrated the direct causal role of Nr4a1 in sustained activation of Cartpt and in attenuation of cocaine-evoked behavior. Our findings provide evidence that targeting abstinence-induced homeostatic gene expression is a potential therapeutic target in cocaine addiction.

RevDate: 2020-04-08
CmpDate: 2020-04-07

Mori L, G De Libero (2020)

'Bohemian Rhapsody' of MR1T cells.

Nature immunology, 21(2):108-110.

RevDate: 2020-04-08
CmpDate: 2020-04-07

Sirvent S, Vallejo AF, Davies J, et al (2020)

Genomic programming of IRF4-expressing human Langerhans cells.

Nature communications, 11(1):313.

Langerhans cells (LC) can prime tolerogenic as well as immunogenic responses in skin, but the genomic states and transcription factors (TF) regulating these context-specific responses are unclear. Bulk and single-cell transcriptional profiling demonstrates that human migratory LCs are robustly programmed for MHC-I and MHC-II antigen presentation. Chromatin analysis reveals enrichment of ETS-IRF and AP1-IRF composite regulatory elements in antigen-presentation genes, coinciding with expression of the TFs, PU.1, IRF4 and BATF3 but not IRF8. Migration of LCs from the epidermis is accompanied by upregulation of IRF4, antigen processing components and co-stimulatory molecules. TNF stimulation augments LC cross-presentation while attenuating IRF4 expression. CRISPR-mediated editing reveals IRF4 to positively regulate the LC activation programme, but repress NF2EL2 and NF-kB pathway genes that promote responsiveness to oxidative stress and inflammatory cytokines. Thus, IRF4-dependent genomic programming of human migratory LCs appears to enable LC maturation while attenuating excessive inflammatory and immunogenic responses in the epidermis.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Ho J, Zhao M, Wojcik S, et al (2020)

The application of the CRISPR-Cas9 system in Pseudomonas syringae pv. actinidiae.

Journal of medical microbiology, 69(3):478-486.

Introduction.Pseudomonas syringae pv. actinidiae (Psa) has emerged as a major bacterial pathogen of kiwifruit cultivation throughout the world.Aim. We aim to introduce a CRISPR-Cas9 system, a commonly used genome editing tool, into Psa. The protocols may also be useful in other Pseudomonas species.Methodology. Using standard molecular biology techniques, we modified plasmid pCas9, which carries the CRISPR-Cas9 sequences from Streptococcus pyogenes, for use in Psa. The final plasmid, pJH1, was produced in a series of steps and is maintained with selection in both Escherichia coli and Psa.Results. We have constructed plasmids carrying a CRISPR-Cas9 system based on that of S. pyogenes, which can be maintained, under selection, in Psa. We have shown that the gene targeting capacity of the CRISPR-Cas9 system is active and that the Cas9 protein is able to cleave the targeted sites. The Cas9 was directed to several different sites in the P. syringae genome. Using Cas9 we have generated Psa transformants that no longer carry the native plasmid present in Psa, and other transformants that lack the integrative, conjugative element, Pac_ICE1. Targeting of a specific gene, a chromosomal non-ribosomal peptide synthetase, led to gene knockouts with the transformants having deletions encompassing the target site.Conclusion. We have constructed shuttle plasmids carrying a CRISPR-Cas9 system that are maintained in both E. coli and P. syringae pv. actinidiae. We have used this gene editing system to eliminate features of the accessory genome (plasmids or ICEs) from Psa and to target a single chromosomal gene.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Anonymous (2020)

When the world needs to know.

Nature biotechnology, 38(1):1.

RevDate: 2020-04-08
CmpDate: 2020-04-07

Alcaraz E, Vilardell J, Borgo C, et al (2020)

Effects of CK2β subunit down-regulation on Akt signalling in HK-2 renal cells.

PloS one, 15(1):e0227340.

The PI3K/Akt pathway is interconnected to protein kinase CK2, which directly phosphorylates Akt1 at S129. We have previously found that, in HK-2 renal cells, downregulation of the CK2 regulatory subunit β (shCK2β cells) reduces S129 Akt phosphorylation. Here, we investigated in more details how the different CK2 isoforms impact on Akt and other signaling pathways. We found that all CK2 isoforms phosphorylate S129 in vitro, independently of CK2β. However, in HK-2 cells the dependence on CK2β was confirmed by rescue experiments (CK2β re-expression in shCK2β HK-2 cells), suggesting the presence of additional components that drive Akt recognition by CK2 in cells. We also found that CK2β downregulation altered the phosphorylation ratio between the two canonical Akt activation sites (pT308 strongly reduced, pS473 slightly increased) in HK-2 cells. Similar results were found in other cell lines where CK2β was stably knocked out by CRISPR-Cas9 technology. The phosphorylation of rpS6 S235/S236, a downstream effector of Akt, was strongly reduced in shCK2β HK-2 cells, while the phosphorylation of two Akt direct targets, PRAS40 T246 and GSK3β S9, was increased. Differently to what observed in response to CK2β down-regulation, the chemical inhibition of CK2 activity by cell treatment with the specific inhibitor CX-4945 reduced both the Akt canonical sites, pT308 and pS473. In CX-4945-treated cells, the changes in rpS6 pS235/S236 and GSK3β pS9 mirrored those induced by CK2β knock-down (reduction and slight increase, respectively); on the contrary, the effect on PRAS40 pT246 phosphorylation was sharply different, being strongly reduced by CK2 inhibition; this suggests that this Akt target might be dependent on Akt pS473 status in HK-2 cells. Since PI3K/Akt and ERK1/2/p90rsk pathways are known to be interconnected and both modulated by CK2, with GSK3β pS9 representing a convergent point, we investigated if ERK1/2/p90rsk signaling was affected by CK2β knock-down and CX-4945 treatment in HK-2 cells. We found that p90rsk was insensitive to any kind of CK2 targeting; therefore, the observation that, similarly, GSK3β pS9 was not reduced by CK2 blockade suggests that GSK3β phosphorylation is mainly under the control of p90rsk in these cells. However, we found that the PI3K inhibitor LY294002 reduced GSK3β pS9, and concomitantly decreased Snail1 levels (a GSK3β target and Epithelial-to-Mesenchymal transition marker). The effects of LY294002 were observed also in CK2β-downregulated cells, suggesting that reducing GSK3β pS9 could be a strategy to control Snail1 levels in any situation where CK2β is defective, as possibly occurring in cancer cells.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Foronda M, Tarumoto Y, Schatoff EM, et al (2019)

Tankyrase inhibition sensitizes cells to CDK4 blockade.

PloS one, 14(12):e0226645.

Tankyrase (TNKS) 1/2 are positive regulators of WNT signaling by controlling the activity of the ß-catenin destruction complex. TNKS inhibitors provide an opportunity to suppress hyperactive WNT signaling in tumors, however, they have shown limited anti-proliferative activity as a monotherapy in human cancer cell lines. Here we perform a kinome-focused CRISPR screen to identify potential effective drug combinations with TNKS inhibition. We show that the loss of CDK4, but not CDK6, synergizes with TNKS1/2 blockade to drive G1 cell cycle arrest and senescence. Through precise modelling of cancer-associated mutations using cytidine base editors, we show that this therapeutic approach is absolutely dependent on suppression of canonical WNT signaling by TNKS inhibitors and is effective in cells from multiple epithelial cancer types. Together, our results suggest that combined WNT and CDK4 inhibition might provide a potential therapeutic strategy for difficult-to-treat epithelial tumors.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Dempster JM, Pacini C, Pantel S, et al (2019)

Agreement between two large pan-cancer CRISPR-Cas9 gene dependency data sets.

Nature communications, 10(1):5817.

Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Lian J, Schultz C, Cao M, et al (2019)

Multi-functional genome-wide CRISPR system for high throughput genotype-phenotype mapping.

Nature communications, 10(1):5794.

Genome-scale engineering is an indispensable tool to understand genome functions due to our limited knowledge of cellular networks. Unfortunately, most existing methods for genome-wide genotype-phenotype mapping are limited to a single mode of genomic alteration, i.e. overexpression, repression, or deletion. Here we report a multi-functional genome-wide CRISPR (MAGIC) system to precisely control the expression level of defined genes to desired levels throughout the whole genome. By combining the tri-functional CRISPR system and array-synthesized oligo pools, MAGIC is used to create, to the best of our knowledge, one of the most comprehensive and diversified genomic libraries in yeast ever reported. The power of MAGIC is demonstrated by the identification of previously uncharacterized genetic determinants of complex phenotypes, particularly those having synergistic interactions when perturbed to different expression levels. MAGIC represents a powerful synthetic biology tool to investigate fundamental biological questions as well as engineer complex phenotypes for biotechnological applications.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Ujihara Y, Kanagawa M, Mohri S, et al (2019)

Elimination of fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.

Nature communications, 10(1):5754.

Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Woronik A, Tunström K, Perry MW, et al (2019)

A transposable element insertion is associated with an alternative life history strategy.

Nature communications, 10(1):5757 pii:10.1038/s41467-019-13596-2.

Tradeoffs affect resource allocation during development and result in fitness consequences that drive the evolution of life history strategies. Yet despite their importance, we know little about the mechanisms underlying life history tradeoffs. Many species of Colias butterflies exhibit an alternative life history strategy (ALHS) where females divert resources from wing pigment synthesis to reproductive and somatic development. Due to this reallocation, a wing color polymorphism is associated with the ALHS: either yellow/orange or white. Here we map the locus associated with this ALHS in Colias crocea to a transposable element insertion located downstream of the Colias homolog of BarH-1, a homeobox transcription factor. Using CRISPR/Cas9 gene editing, antibody staining, and electron microscopy we find white-specific expression of BarH-1 suppresses the formation of pigment granules in wing scales and gives rise to white wing color. Lipid and transcriptome analyses reveal physiological differences associated with the ALHS. Together, these findings characterize a mechanism for a female-limited ALHS.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Lentsch E, Li L, Pfeffer S, et al (2019)

CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines.

International journal of molecular sciences, 20(22):.

In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Xiong ZQ, Wei YY, Kong LH, et al (2020)

Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis.

Journal of dairy science, 103(1):161-165.

Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter Pnisin was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P44 drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Chiarella AM, Butler KV, Gryder BE, et al (2020)

Dose-dependent activation of gene expression is achieved using CRISPR and small molecules that recruit endogenous chromatin machinery.

Nature biotechnology, 38(1):50-55.

Gene expression can be activated or suppressed using CRISPR--Cas9 systems. However, tools that enable dose-dependent activation of gene expression without the use of exogenous transcription regulatory proteins are lacking. Here we describe chemical epigenetic modifiers (CEMs) designed to activate the expression of target genes by recruiting components of the endogenous chromatin-activating machinery, eliminating the need for exogenous transcriptional activators. The system has two parts: catalytically inactive Cas9 (dCas9) in complex with FK506-binding protein (FKBP) and a CEM consisting of FK506 linked to a molecule that interacts with cellular epigenetic machinery. We show that CEMs upregulate gene expression at target endogenous loci up to 20-fold or more depending on the gene. We also demonstrate dose-dependent control of transcriptional activation, function across multiple diverse genes, reversibility of CEM activity and specificity of our best-in-class CEM across the genome.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Zheng G, Zhu Q, Dong J, et al (2019)

Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects.

BMC biotechnology, 19(1):74.

BACKGROUND: Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes.

RESULTS: Here, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently.

CONCLUSIONS: A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Chan B (2020)

Improving the justice-based argument for conducting human gene editing research to cure sickle cell disease.

Bioethics, 34(2):200-202.

In a recent article, Marilyn Baffoe-Bonnie offers three arguments that conducting CRISPR/Cas9 biotechnology research to cure sickle cell disease (SCD) would help address historical and current injustices in SCD research and care. I will grant that the first argument is sound, but show that the second and third arguments suffer from roughly the same defect, which is that they really argue for something else rather than for conducting CRISPR/Cas9 research to cure SCD. I conclude that a better justice-based argument would use only Baffoe-Bonnie's first argument.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Arndell T, Sharma N, Langridge P, et al (2019)

gRNA validation for wheat genome editing with the CRISPR-Cas9 system.

BMC biotechnology, 19(1):71.

BACKGROUND: The CRISPR-Cas9 system is a powerful and versatile tool for crop genome editing. However, achieving highly efficient and specific editing in polyploid species can be a challenge. The efficiency and specificity of the CRISPR-Cas9 system depends critically on the gRNA used. Here, we assessed the activities and specificities of seven gRNAs targeting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in hexaploid wheat protoplasts. EPSPS is the biological target of the widely used herbicide glyphosate.

RESULTS: The seven gRNAs differed substantially in their on-target activities, with mean indel frequencies ranging from 0% to approximately 20%. There was no obvious correlation between experimentally determined and in silico predicted on-target gRNA activity. The presence of a single mismatch within the seed region of the guide sequence greatly reduced but did not abolish gRNA activity, whereas the presence of an additional mismatch, or the absence of a PAM, all but abolished gRNA activity. Large insertions (≥20 bp) of DNA vector-derived sequence were detected at frequencies up to 8.5% of total indels. One of the gRNAs exhibited several properties that make it potentially suitable for the development of non-transgenic glyphosate resistant wheat.

CONCLUSIONS: We have established a rapid and reliable method for gRNA validation in hexaploid wheat protoplasts. The method can be used to identify gRNAs that have favourable properties. Our approach is particularly suited to polyploid species, but should be applicable to any plant species amenable to protoplast transformation.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Jain PK, Lo JH, Rananaware S, et al (2019)

Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes.

Nanoscale, 11(44):21317-21323.

There is a critical need for the development of safe and efficient delivery technologies for CRISPR/Cas9 to advance translation of genome editing to the clinic. Non-viral methods that are simple, efficient, and completely based on biologically-derived materials could offer such potential. Here we report a simple and modular tandem peptide-based nanocomplex system with cell-targeting capacity that efficiently combines guide RNA (sgRNA) with Cas9 protein, and facilitates internalization of sgRNA/Cas9 ribonucleoprotein complexes to yield robust genome editing across multiple cell lines.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Kawasaki K, Fujii M, Sugimoto S, et al (2020)

Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma.

Gastroenterology, 158(3):638-651.e8.

BACKGROUND & AIMS: Traditional serrated adenomas (TSAs) are rare colorectal polyps with unique histologic features. Fusions in R-spondin genes have been found in TSAs, but it is not clear whether these are sufficient for TSA development, due to the lack of a chromosome engineering platform for human tissues. We studied the effects of fusions in R-spondin genes and other genetic alterations found in TSA using CRISPR-Cas9-mediated chromosome and genetic modification of human colonic organoids.

METHODS: We introduced chromosome rearrangements that involve R-spondin genes into human colonic organoids, with or without disruption of TP53, using CRISPR-Cas9 (chromosome-engineered organoids). We then knocked a mutation into BRAF encoding the V600E substitution and overexpressed the GREM1 transgene; the organoids were transplanted into colons of NOG mice and growth of xenograft tumors was measured. Colon tissues were collected and analyzed by immunohistochemistry or in situ hybridization. We also established 2 patient-derived TSA organoid lines and characterized their genetic features and phenotypes. We inserted a bicistronic cassette expressing a dimerizer-inducible suicide gene and fluorescent marker downstream of the LGR5 gene in the chromosome-engineered organoids; addition of the dimerizer eradicates LGR5+ cells. Some tumor-bearing mice were given intraperitoneal injections of the dimerizer to remove LGR5-expressing cells.

RESULTS: Chromosome engineering of organoids required disruption of TP53 or culture in medium containing IGF1 and FGF2. In colons of mice, organoids that expressed BRAFV600E and fusions in R-spondin genes formed flat serrated lesions. Patient-derived TSA organoids grew independent of exogenous R-spondin, and 1 line grew independent of Noggin. Organoids that overexpressed GREM1, in addition to BRAFV600E and fusions in R-spondin genes, formed polypoid tumors in mice that had histologic features similar to TSAs. Xenograft tumors persisted after loss of LGR5-expressing cells.

CONCLUSIONS: We demonstrated efficient chromosomal engineering of human normal colon organoids. We introduced genetic and chromosome alterations into human colon organoids found in human TSAs; tumors grown from these organoids in mice had histopathology features of TSAs. This model might be used to study progression of human colorectal tumors with RSPO fusion gene and GREM1 overexpression.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Fan Y, Li J, Wei W, et al (2019)

Ku80 gene knockdown by the CRISPR/Cas9 technique affects the biological functions of human thyroid carcinoma cells.

Oncology reports, 42(6):2486-2498.

In the present study, to evaluate the role of Ku80 in thyroid carcinoma (TC), 86 thyroid tissue samples from patients with a spectrum of thyroid disorders were examined for protein levels of Ku80, nuclear factor‑κB (NF‑κB) and RET/TC by immunohistochemistry. Furthermore, in TC cells, Ku80 mRNA was detected by reverse transcription‑quantitative PCR analysis and silenced using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‑associated protein 9 (Cas9) technique to assess its role. An antibody array was used to identify Ku80‑related regulatory genes. The protein levels of Ku80 in the TC tissues were significantly higher than those in non‑neoplastic adjacent tissue samples (P<0.01). The activation of NF‑kB and expression of RET/TC in the TC group were significantly increased (P<0.05) and were correlated with the protein expression of Ku80 (P<0.05). In papillary TC cells, the mRNA levels of Ku80 were high; Ku80 knockdown resulted in reductions in proliferation, invasion and colony formation, increased apoptosis, and reduced levels of proteins involved in MAPK signaling, cell proliferation and apoptosis. The high expression of Ku80 in TC was found to be associated with the expression of RET/TC and activation of NF‑κB, and Ku80 knockdown decreased the malignancy of TC cells.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Tong Y, Whitford CM, Robertsen HL, et al (2019)

Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST.

Proceedings of the National Academy of Sciences of the United States of America, 116(41):20366-20375.

Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide-resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Binger KJ, Neukam M, Tattikota SG, et al (2019)

Atp6ap2 deletion causes extensive vacuolation that consumes the insulin content of pancreatic β cells.

Proceedings of the National Academy of Sciences of the United States of America, 116(40):19983-19988.

Pancreatic β cells store insulin within secretory granules which undergo exocytosis upon elevation of blood glucose levels. Crinophagy and autophagy are instead responsible to deliver damaged or old granules to acidic lysosomes for intracellular degradation. However, excessive consumption of insulin granules can impair β cell function and cause diabetes. Atp6ap2 is an essential accessory component of the vacuolar ATPase required for lysosomal degradative functions and autophagy. Here, we show that Cre recombinase-mediated conditional deletion of Atp6ap2 in mouse β cells causes a dramatic accumulation of large, multigranular vacuoles in the cytoplasm, with reduction of insulin content and compromised glucose homeostasis. Loss of insulin stores and gigantic vacuoles were also observed in cultured insulinoma INS-1 cells upon CRISPR/Cas9-mediated removal of Atp6ap2. Remarkably, these phenotypic alterations could not be attributed to a deficiency in autophagy or acidification of lysosomes. Together, these data indicate that Atp6ap2 is critical for regulating the stored insulin pool and that a balanced regulation of granule turnover is key to maintaining β cell function and diabetes prevention.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Fujihara Y, Noda T, Kobayashi K, et al (2019)

Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice.

Proceedings of the National Academy of Sciences of the United States of America, 116(37):18498-18506.

CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. Although Gdpd1 and Gdpd4 mutant mice were fertile, disruptions of Cst and Pate gene clusters and Lypd4 resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Wang L, Zhao L, Zhang X, et al (2019)

Large-scale identification and functional analysis of NLR genes in blast resistance in the Tetep rice genome sequence.

Proceedings of the National Academy of Sciences of the United States of America, 116(37):18479-18487.

Tetep is a rice cultivar known for broad-spectrum resistance to blast, a devastating fungal disease. The molecular basis for its broad-spectrum resistance is still poorly understood. Is it because Tetep has many more NLR genes than other cultivars? Or does Tetep possess multiple major NLR genes that can individually confer broad-spectrum resistance to blast? Moreover, are there many interacting NLR pairs in the Tetep genome? We sequenced its genome, obtained a high-quality assembly, and annotated 455 nucleotide-binding site leucine-rich repeat (NLR) genes. We cloned and tested 219 NLR genes as transgenes in 2 susceptible cultivars using 5 to 12 diversified pathogen strains; in many cases, fewer than 12 strains were successfully cultured for testing. Ninety cloned NLRs showed resistance to 1 or more pathogen strains and each strain was recognized by multiple NLRs. However, few NLRs showed resistance to >6 strains, so multiple NLRs are apparently required for Tetep's broad-spectrum resistance to blast. This was further supported by the pedigree analyses, which suggested a correlation between resistance and the number of Tetep-derived NLRs. In developing a method to identify NLR pairs each of which functions as a unit, we found that >20% of the NLRs in the Tetep and 3 other rice genomes are paired. Finally, we designed an extensive set of molecular markers for rapidly introducing clustered and paired NLRs in the Tetep genome for breeding new resistant cultivars. This study increased our understanding of the genetic basis of broad-spectrum blast resistance in rice.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Nakagawa Y, T Kaneko (2019)

Rapid and efficient production of genome-edited animals by electroporation into oocytes injected with frozen or freeze-dried sperm.

Cryobiology, 90:71-74.

Sperm preservation is a useful technique for maintaining valuable animal strains. Rat sperm could be frozen or freeze-dried in a simple Tris-EDTA solution (TE buffer), and oocytes that were fertilized with these sperm by intracytoplasmic sperm injection (ICSI) developed into offspring. Genome editing with the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system enables the rapid production of genetically modified rats. The recent innovative method, named the TAKE method, could easily produce genome edited rats by electroporation of endonucleases into embryos. Although various rat strains have been applied for genome editing, genome editing using strains that were preserved as sperm took longer because it required collecting embryos after maturation of animals regenerated from sperm. To reduce the production period, we directly electroporated Cas9 protein and gRNA into oocytes that were injected with frozen or freeze-dried sperm in TE buffer. No effect of electroporation until 30 V to ICSI-embryos derived from frozen or freeze-dried sperm were shown in the development of offspring. Furthermore, the rate of genome editing in offspring was high (56% for frozen and 50% for freeze-dried sperm). These results concluded that the combination of ICSI and the TAKE method was useful for the rapid production of genome-edited animals from sperm that have been preserved as genetic resources.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Yuan F, Zhou J, Xu L, et al (2019)

GABA receptors differentially regulate life span and health span in C. elegans through distinct downstream mechanisms.

American journal of physiology. Cell physiology, 317(5):C953-C963.

GABA, a prominent inhibitory neurotransmitter, is best known to regulate neuronal functions in the nervous system. However, much less is known about the role of GABA signaling in other physiological processes. Interestingly, recent work showed that GABA signaling can regulate life span via a metabotropic GABAB receptor in Caenorhabditis elegans. However, the role of other types of GABA receptors in life span has not been clearly defined. It is also unclear whether GABA signaling regulates health span. Here, using C. elegans as a model, we systematically interrogated the role of various GABA receptors in both life span and health span. We find that mutations in four different GABA receptors extend health span by promoting resistance to stress and pathogen infection and that two such receptor mutants also show extended life span. Different GABA receptors engage distinct transcriptional factors to regulate life span and health span, and even the same receptor regulates life span and health span via different transcription factors. Our results uncover a novel, profound role of GABA signaling in aging in C. elegans, which is mediated by different GABA receptors coupled to distinct downstream effectors.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Tian R, Gachechiladze MA, Ludwig CH, et al (2019)

CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons.

Neuron, 104(2):239-255.e12.

CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer cell lines rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic screens in human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes in such neurons and present results from three complementary genetic screens. First, a survival-based screen revealed neuron-specific essential genes and genes that improved neuronal survival upon knockdown. Second, a screen with a single-cell transcriptomic readout uncovered several examples of genes whose knockdown had strikingly cell-type-specific consequences. Third, a longitudinal imaging screen detected distinct consequences of gene knockdown on neuronal morphology. Our results highlight the power of unbiased genetic screens in iPSC-derived differentiated cell types and provide a platform for systematic interrogation of normal and disease states of neurons. VIDEO ABSTRACT.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Kwart D, Gregg A, Scheckel C, et al (2019)

A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP β-CTFs, Not Aβ.

Neuron, 104(2):256-270.e5.

Familial Alzheimer's disease (fAD) results from mutations in the amyloid precursor protein (APP) and presenilin (PSEN1 and PSEN2) genes. Here we leveraged recent advances in induced pluripotent stem cell (iPSC) and CRISPR/Cas9 genome editing technologies to generate a panel of isogenic knockin human iPSC lines carrying APP and/or PSEN1 mutations. Global transcriptomic and translatomic profiling revealed that fAD mutations have overlapping effects on the expression of AD-related and endocytosis-associated genes. Mutant neurons also increased Rab5+ early endosome size. APP and PSEN1 mutations had discordant effects on Aβ production but similar effects on APP β C-terminal fragments (β-CTFs), which accumulate in all mutant neurons. Importantly, endosomal dysfunction correlated with accumulation of β-CTFs, not Aβ, and could be rescued by pharmacological modulation of β-secretase (BACE). These data display the utility of our mutant iPSCs in studying AD-related phenotypes in a non-overexpression human-based system and support mounting evidence that β-CTF may be critical in AD pathogenesis.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Ochiai H (2019)

Real-Time Observation of Localization and Expression (ROLEX) System for Live Imaging of the Transcriptional Activity and Nuclear Position of a Specific Endogenous Gene.

Methods in molecular biology (Clifton, N.J.), 2038:35-45.

Long genomic DNA is folded in a cell-type-specific manner and stored in the cell nucleus. The higher-order structure of genomic DNA is thought to be important for DNA transcription, repair, and replication. Recent advancements in live cell imaging techniques that enable the labeling of specific genomic loci and RNA have made it possible to capture the dynamic relationships between higher-order genomic structure and gene expression. We have established the real-time observation of localization and expression (ROLEX) system for live imaging of the transcriptional state and nuclear position of a specific endogenous gene. In this chapter, I will introduce the detailed protocol of ROLEX imaging in mouse embryonic stem cells.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Garcia-Marques J, Yang CP, Espinosa-Medina I, et al (2019)

Unlimited Genetic Switches for Cell-Type-Specific Manipulation.

Neuron, 104(2):227-238.e7.

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

RevDate: 2020-04-08
CmpDate: 2020-04-08

Readler JM, AlKahlout AS, Sharma P, et al (2019)

Isoform specific editing of the coxsackievirus and adenovirus receptor.

Virology, 536:20-26.

The Coxsackievirus and adenovirus receptor (CAR) is both a viral receptor and cell adhesion protein. CAR has two transmembrane isoforms that localize distinctly in polarized epithelial cells. Whereas the seven exon-encoded isoform (CAREx7) exhibits basolateral localization, the eight exon-encoded isoform (CAREx8) can localize to the apical epithelial surface where it can mediate luminal adenovirus infection. To further understand the distinct biological functions of these two isoforms, CRISPR/Cas9 genomic editing was used to specifically delete the eighth exon of the CXADR gene in a Madine Darby Canine Kidney (MDCK) cell line with a stably integrated lentiviral doxycycline-inducible CAREx8 cDNA. The gene-edited clone demonstrated a significant reduction in adenovirus susceptibility when both partially and fully polarized, and doxycycline-induction of CAREx8 restored sensitivity to adenovirus. These data reinforce the importance of CAREx8 in apical adenovirus infection and provide a new model cell line to probe isoform specific biological functions of CAR.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Li X, Hao F, Hu X, et al (2019)

Generation of Tβ4 knock-in Cashmere goat using CRISPR/Cas9.

International journal of biological sciences, 15(8):1743-1754.

The cashmere goat breed is known to provide excellent quality cashmere. Here, we attempted to breed high-yielding cashmere goats by specifically inserting the Tβ4 gene into the goat CCR5 locus and provided an animal model for future research. We successfully obtained Tβ4 knock-in goat without any screening and fluorescent markers using CRISPR/Cas9 technology. A series of experiments were performed to examine physical conditions and characteristics of the Tβ4 knock-in goat. The goat exhibited an increase in cashmere yield by 74.5% without affecting the fineness and quality. Additionally, RNA-seq analysis indicated that Tβ4 may promote hair growth by affecting processes such as vasoconstriction, angiogenesis, and vascular permeability around secondary hair follicles. Together, our study can significantly improve the breeding of cashmere goat and thereby increase economic efficiency.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Khoshnejad M, Brenner JS, Parhiz H, et al (2019)

CRISPR/Cas9-Mediated Genetic Engineering of Hybridomas for Creation of Antibodies that Allow for Site-Specific Conjugation.

Methods in molecular biology (Clifton, N.J.), 2033:81-93.

Covalent conjugation of chemical moieties to antibodies has numerous applications, including antibody-drug conjugates, antibody conjugation for diagnostics, and more. Most nonspecific chemical conjugation methods ligate onto any of a number of sites on the antibody, leading to multiple conjugated species, many of which perturb antibody function. To solve these problems, we used CRISPR/Cas9-edited hybridomas to introduce a Sortase tag (LPXTG) and a Flag tag at the 3' end of the CH3 heavy chain region of a mouse monoclonal antibody. The Flag tag allows easy purification of the antibody, while the LPXTG is then acted on by the bacterial transpeptidase Sortase to site-specifically add on any of a number of chemical moieties that possess a triglycine repeat. This technique thus allows rapid production of an antibody onto which a wide array of chemical moieties can be site-specifically conjugated.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Panganiban RA, Park HR, Sun M, et al (2019)

Genome-wide CRISPR screen identifies suppressors of endoplasmic reticulum stress-induced apoptosis.

Proceedings of the National Academy of Sciences of the United States of America, 116(27):13384-13393.

Sensing misfolded proteins in the endoplasmic reticulum (ER), cells initiate the ER stress response and, when overwhelmed, undergo apoptosis. However, little is known about how cells prevent excessive ER stress response and cell death to restore homeostasis. Here, we report the identification and characterization of cellular suppressors of ER stress-induced apoptosis. Using a genome-wide CRISPR library, we screen for genes whose inactivation further increases ER stress-induced up-regulation of C/EBP homologous protein 10 (CHOP)-the transcription factor central to ER stress-associated apoptosis. Among the top validated hits are two interacting components of the polycomb repressive complex (L3MBTL2 [L(3)Mbt-Like 2] and MGA [MAX gene associated]), and microRNA-124-3 (miR-124-3). CRISPR knockout of these genes increases CHOP expression and sensitizes cells to apoptosis induced by multiple ER stressors, while overexpression confers the opposite effects. L3MBTL2 associates with the CHOP promoter in unstressed cells to repress CHOP induction but dissociates from the promoter in the presence of ER stress, whereas miR-124-3 directly targets the IRE1 branch of the ER stress pathway. Our study reveals distinct mechanisms that suppress ER stress-induced apoptosis and may lead to a better understanding of diseases whose pathogenesis is linked to overactive ER stress response.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Boned Del Río I, Young LC, Sari S, et al (2019)

SHOC2 complex-driven RAF dimerization selectively contributes to ERK pathway dynamics.

Proceedings of the National Academy of Sciences of the United States of America, 116(27):13330-13339.

Despite the crucial role of RAF kinases in cell signaling and disease, we still lack a complete understanding of their regulation. Heterodimerization of RAF kinases as well as dephosphorylation of a conserved "S259" inhibitory site are important steps for RAF activation but the precise mechanisms and dynamics remain unclear. A ternary complex comprised of SHOC2, MRAS, and PP1 (SHOC2 complex) functions as a RAF S259 holophosphatase and gain-of-function mutations in SHOC2, MRAS, and PP1 that promote complex formation are found in Noonan syndrome. Here we show that SHOC2 complex-mediated S259 RAF dephosphorylation is critically required for growth factor-induced RAF heterodimerization as well as for MEK dissociation from BRAF. We also uncover SHOC2-independent mechanisms of RAF and ERK pathway activation that rely on N-region phosphorylation of CRAF. In DLD-1 cells stimulated with EGF, SHOC2 function is essential for a rapid transient phase of ERK activation, but is not required for a slow, sustained phase that is instead driven by palmitoylated H/N-RAS proteins and CRAF. Whereas redundant SHOC2-dependent and -independent mechanisms of RAF and ERK activation make SHOC2 dispensable for proliferation in 2D, KRAS mutant cells preferentially rely on SHOC2 for ERK signaling under anchorage-independent conditions. Our study highlights a context-dependent contribution of SHOC2 to ERK pathway dynamics that is preferentially engaged by KRAS oncogenic signaling and provides a biochemical framework for selective ERK pathway inhibition by targeting the SHOC2 holophosphatase.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Lee J, Ma J, K Lee (2019)

Direct delivery of adenoviral CRISPR/Cas9 vector into the blastoderm for generation of targeted gene knockout in quail.

Proceedings of the National Academy of Sciences of the United States of America, 116(27):13288-13292.

Zygotes at the 1-cell stage have been genetically modified by microinjecting the CRISPR/Cas9 components for the generation of targeted gene knockout in mammals. In the avian species, genetic modification of the zygote is difficult because its unique reproductive system limits the accessibility of the zygote at the 1-cell stage. To date, only a few CRISPR/Cas9-mediated gene knockouts have been reported using the chicken as a model among avian species, which requires 3 major processes: isolation and culture of primordial germ cells (PGCs), modification of the genome of PGCs in vitro, and injection of the PGCs into the extraembryonic blood vessel at the early embryonic stages when endogenous PGCs migrate through circulation to the genital ridge. In the present study, the adenoviral CRISPR/Cas9 vector was directly injected into the quail blastoderm in newly laid eggs. The resulting chimeras generated offspring with targeted mutations in the melanophilin (MLPH) gene, which is involved in melanosome transportation and feather pigmentation. MLPH homozygous mutant quail exhibited gray plumage, whereas MLPH heterozygous mutants and wild-type quail exhibited dark brown plumage. In addition, the adenoviral vector was not integrated into the genome of knockout quail, and no mutations were detected in potential off-target regions. This method of generating genome-edited poultry is expected to accelerate avian research and has potential applications for developing superior genetic lines for poultry production in the industry.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Goto N, Fukuda A, Yamaga Y, et al (2019)

Lineage tracing and targeting of IL17RB+ tuft cell-like human colorectal cancer stem cells.

Proceedings of the National Academy of Sciences of the United States of America, 116(26):12996-13005.

Cancer stem cell (CSC)-specific markers may be potential therapeutic targets. We previously identified that Dclk1, a tuft cell marker, marks tumor stem cells (TSCs) in mouse intestinal adenomas. Based on the analysis of mouse Dclk1+ tumor cells, we aimed to identify a CSC-specific cell surface marker in human colorectal cancers (hCRCs) and validate the therapeutic effect of targeting it. IL17RB was distinctively expressed by Dclk1+ mouse intestinal tumor cells. Using Il17rb-CreERT2-IRES-EGFP mice, we show that IL17RB marked intestinal TSCs in an IL13-dependent manner. Tuft cell-like cancer cells were detected in a subset of hCRCs. In these hCRCs, lineage-tracing experiments in CRISPR-Cas9-mediated IL17RB-CreERT2 knockin organoids and xenograft tumors revealed that IL17RB marks CSCs that expand independently of IL-13. We observed up-regulation of POU2F3, a master regulator of tuft cell differentiation, and autonomous tuft cell-like cancer cell differentiation in the hCRCs. Furthermore, long-term ablation of IL17RB-expressing CSCs strongly suppressed the tumor growth in vivo. These findings reveal insights into a CSC-specific marker IL17RB in a subset of hCRCs, and preclinically validate IL17RB+ CSCs as a cancer therapeutic target.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Wang C, Lu T, Emanuel G, et al (2019)

Imaging-based pooled CRISPR screening reveals regulators of lncRNA localization.

Proceedings of the National Academy of Sciences of the United States of America, 116(22):10842-10851.

Pooled-library CRISPR screening provides a powerful means to discover genetic factors involved in cellular processes in a high-throughput manner. However, the phenotypes accessible to pooled-library screening are limited. Complex phenotypes, such as cellular morphology and subcellular molecular organization, as well as their dynamics, require imaging-based readout and are currently beyond the reach of pooled-library CRISPR screening. Here we report an all imaging-based pooled-library CRISPR screening approach that combines high-content phenotype imaging with high-throughput single guide RNA (sgRNA) identification in individual cells. In this approach, sgRNAs are codelivered to cells with corresponding barcodes placed at the 3' untranslated region of a reporter gene using a lentiviral delivery system with reduced recombination-induced sgRNA-barcode mispairing. Multiplexed error-robust fluorescence in situ hybridization (MERFISH) is used to read out the barcodes and hence identify the sgRNAs with high accuracy. We used this approach to screen 162 sgRNAs targeting 54 RNA-binding proteins for their effects on RNA localization to nuclear compartments and uncovered previously unknown regulatory factors for nuclear RNA localization. Notably, our screen revealed both positive and negative regulators for the nuclear speckle localization of a long noncoding RNA, MALAT1, suggesting a dynamic regulation of lncRNA localization in subcellular compartments.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Eom SY, Hwang SH, Yeom H, et al (2019)

An ATG5 knockout promotes paclitaxel resistance in v-Ha-ras-transformed NIH 3T3 cells.

Biochemical and biophysical research communications, 513(1):234-241.

Autophagy plays a contradictory role in cell survival and death. Here, we investigated changes in paclitaxel sensitivity of cells with an ATG5 gene-knockout (KO), incapable of synthesizing an E3 ubiquitin ligase necessary for autophagy. The ATG5 KO in v-Ha-ras-transformed NIH 3T3 cells (Ras-NIH 3T3) was established using the CRISPR/Cas9 system. An LC3 immunoblot and a qRT-PCR assay were used to confirm the KO of functional ATG5. We found that the ATG5 KO led to paclitaxel resistance in Ras-NIH 3T3 cells through an ATP-binding cassette (ABC) transporter-independent mechanism. Flow cytometric analyses revealed that paclitaxel induced a remarkable significant G2/M arrest in parental cells, whereas it was relatively less effective in ATG5 KO cells. Additionally, the proportion of early apoptotic cells significantly decreased in ATG5 KO cells treated with paclitaxel than in parental cells. Interestingly, overexpression of ATG5 N-terminal cleavage product in ATG5 KO cells restored their sensitivity to paclitaxel. Taken together, our results suggest that ATG5 KO cells are resistant to paclitaxel due to the inability to produce tATG5.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Ali M, Mutahir Z, A Riaz (2019)

CRISPR/Cas9 engineering of ERK5 identifies its FAK/PYK2 dependent role in adhesion-mediated cell survival.

Biochemical and biophysical research communications, 513(1):179-185.

Extracellular signal-regulated kinase 5 (ERK5) is now considered a key regulator of breast cancer cell proliferation, migration and invasion. It is also implicated in growth factor induced anti-apoptotic signaling. But its contribution to adhesion-induced survival signaling is not clear. In the present study, using CRISPR/Cas9 editing, we knocked-out ERK5 expression in several cancer cell lines. Then MDA-MB 231 breast cancer cells lacking ERK5 were used to understand its role in adhesion-mediated cell viability. We demonstrated that ERK5 deficient cells exhibited reduced cell attachment to matrix proteins fibronectin and vitronectin. The adhesion ability of these cells was further reduced upon chemical inhibition of focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2) by PF 431396. FAK/PYK2 inhibited ERK5 knock-out cells also showed markedly reduced cell-viability and increased apoptotic signaling. This was evident from the detection of cleaved PARP and caspase 9 in these cells. Thus, our data suggests a FAK/PYK2 regulated pro-survival role of ERK5 in response to cell adhesion.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Kawaguchi K, Sato T, Kondo S, et al (2019)

Stability of the transamidase complex catalyzing GPI anchoring of proteins.

Biochemical and biophysical research communications, 512(3):584-590.

Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors some proteins to the plasma membrane. This anchoring is catalyzed by a transamidase complex (TAC) composed of five subunits: PIG-K, GAA1, PIG-U, PIG-T, and PIG-S (Fig. 1A). PIG-K and GAA1 are predicted to catalyze the first and second steps during attachment of proproteins of GPI-anchored proteins (GPI-APs) to GPI. GPI may be delivered by PIG-U, and PIG-T is required for stability of all TAC subunits when overexpressed in cultured cells. However, protein stability of TAC has not been analyzed using loss-of-function mutants for each subunit. Herein, we analyzed the stability of TAC in knockout and/or knockdown mutants for each subunit. PIG-T and PIG-U, or PIG-T and GAA1, were mutually required for stability, and all three subunits were stable without PIG-S or PIG-K. However, these three subunits were essential for the stability of both PIG-S and PIG-K. By contrast, loss of PIG-S reduced the stability of PIG-K and left the other subunits unaffected. Reduction of PIG-K did not impact any of the other subunits. Thus, PIG-T, PIG-U, and GAA1 may form a core complex associated by PIG-S, and these four subunits may stabilize PIG-K, triggering GPI anchoring reactions. Instability of PIG-K in the absence of the other four subunits may ensure that GPI anchoring is catalyzed only by the completely assembled complex.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Kasamatsu A, Uzawa K, Hayashi F, et al (2019)

Deficiency of lysyl hydroxylase 2 in mice causes systemic endoplasmic reticulum stress leading to early embryonic lethality.

Biochemical and biophysical research communications, 512(3):486-491.

Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2-/-) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2-/- mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Kowalski PS, Rudra A, Miao L, et al (2019)

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery.

Molecular therapy : the journal of the American Society of Gene Therapy, 27(4):710-728.

mRNA has broad potential as a therapeutic. Current clinical efforts are focused on vaccination, protein replacement therapies, and treatment of genetic diseases. The clinical translation of mRNA therapeutics has been made possible through advances in the design of mRNA manufacturing and intracellular delivery methods. However, broad application of mRNA is still limited by the need for improved delivery systems. In this review, we discuss the challenges for clinical translation of mRNA-based therapeutics, with an emphasis on recent advances in biomaterials and delivery strategies, and we present an overview of the applications of mRNA-based delivery for protein therapy, gene editing, and vaccination.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Li R, Qiu Z, Wang X, et al (2019)

Pooled CRISPR/Cas9 reveals redundant roles of plastidial phosphoglycerate kinases in carbon fixation and metabolism.

The Plant journal : for cell and molecular biology, 98(6):1078-1089.

Phosphoglycerate kinase (PGK) is a highly conserved reversible enzyme that participates in both glycolysis and photosynthesis. In Arabidopsis thaliana, one cytosolic PGK (PGKc) and two plastidial PGKs (PGKp) are known. It remains debatable whether the two PGKp isozymes are functionally redundant or specialized in plastidial carbon metabolism and fixation. Here, using a pooled clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) strategy, we found that plants with single mutations in pgkp1 or pgkp2 were not significantly affected, whereas a pgkp1pgkp2 double mutation was lethal due to retarded carbon fixation, suggesting that PGKp isozymes play redundant functional roles. Metabolomic analysis demonstrated that the sugar-deficient pgkp1pgkp2 double mutation was partially complemented by exogenous sugar, although respiration intermediates were not rescued. Chloroplast development was defective in pgkp1pgkp2, due to a deficiency in glycolysis-dependent galactoglycerolipid biosynthesis. Ectopic expression of a plastid targeting PGKc did not reverse the pgkp1pgkp2 double-mutant phenotypes. Therefore, PGKp1 and PGKp2 play redundant roles in carbon fixation and metabolism, whereas the molecular function of PGKc is more divergent. Our study demonstrated the functional conservation and divergence of glycolytic enzymes.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Ji CH, Kim H, HS Kang (2019)

Synthetic Inducible Regulatory Systems Optimized for the Modulation of Secondary Metabolite Production in Streptomyces.

ACS synthetic biology, 8(3):577-586.

Biosynthesis of secondary metabolites is a highly complex process that often requires tight control of their production, as overproduction of metabolites could be toxic and also may cause metabolic burden to their hosts. Tight control of metabolite production could be achieved by expressing key biosynthetic genes under control of an inducible regulatory system. In this study, we employed the modular design approach to build a high performance synthetic inducible regulatory system that displays a large dynamic range and thus is well-suited for the modulation of secondary metabolite production in Streptomyces. To this end, an inducible regulatory system was divided into three separate functional modules: (1) the induction module, (2) the target expression module, and (3) the repressor expression module. Then, these three separate modules were individually optimized in a stepwise manner and assembled to a new system. First, the cumate (CMT) induction module was chosen as the best performing induction module based on the large dynamic range and moderate inducer sensitivity. Then the CMT induction module maintained its performance when combined with diverse constitutive target expression modules, in which overall dynamic ranges varied depending on maximum promoter strengths. Lastly, the repressor expression module was optimized to achieve complete elimination of leaky expression, further increasing the dynamic range of the system. We also demonstrate that any strong constitutive regulatory system could be converted into an inducible regulatory system by simple CRISPR/Cas9-aided markerless insertion of an operator sequence whenever tight control of gene expression is required. We believe that the synthetic inducible regulatory system we report here would become a useful tool in modulating secondary metabolite production in Streptomyces.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Zhang HX, Zhang Y, H Yin (2019)

Genome Editing with mRNA Encoding ZFN, TALEN, and Cas9.

Molecular therapy : the journal of the American Society of Gene Therapy, 27(4):735-746.

Genome-editing technologies based on programmable nucleases have significantly broadened our ability to make precise and direct changes in the genomic DNA of various species, including human cells. Delivery of programmable nucleases into the target tissue or cell is one of the pressing challenges in transforming the technology into medicine. In vitro-transcribed (IVT) mRNA-mediated delivery of nucleases has several advantages, such as transient expression with efficient in vivo and in vitro delivery, no genomic integration, a potentially low off-target rate, and high editing efficiency. This review focuses on key barriers related to IVT mRNA delivery, on developed modes of delivery, and on the application and future prospects of mRNA encoding nuclease-mediated genome editing in research and clinical trials.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Miyamoto T, Takada R, Tobimatsu Y, et al (2019)

OsMYB108 loss-of-function enriches p-coumaroylated and tricin lignin units in rice cell walls.

The Plant journal : for cell and molecular biology, 98(6):975-987.

Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Guo Y, Bao C, Ma D, et al (2019)

Network-Based Combinatorial CRISPR-Cas9 Screens Identify Synergistic Modules in Human Cells.

ACS synthetic biology, 8(3):482-490.

Tumorigenesis is a complex process that is driven by a combination of networks of genes and environmental factors; however, efficient approaches to identifying functional networks that are perturbed by the process of tumorigenesis are lacking. In this study, we provide a comprehensive network-based strategy for the systematic discovery of functional synergistic modules that are causal determinants of inflammation-induced tumorigenesis. Our approach prioritizes candidate genes selected by integrating clinical-based and network-based genome-wide gene prediction methods and identifies functional synergistic modules based on combinatorial CRISPR-Cas9 screening. On the basis of candidate genes inferred de novo from experimental and computational methods to be involved in inflammation and cancer, we used an existing TGFβ1-induced cellular transformation model in colonic epithelial cells and a new combinatorial CRISPR-Cas9 screening strategy to construct an inflammation-induced differential genetic interaction network. The inflammation-induced differential genetic interaction network that we generated yielded functional insights into the genes and functional module combinations, and showed varied responses to the inflammation agents as well as active traditional Chinese medicine compounds. We identified opposing differential genetic interactions of inflammation-induced tumorigenesis: synergistic promotion and suppression. The synergistic promotion state was primarily caused by deletions in the immune and metabolism modules; the synergistic suppression state was primarily induced by deletions in the proliferation and immune modules or in the proliferation and metabolism modules. These results provide insight into possible early combinational targets and biomarkers for inflammation-induced tumorigenesis and highlight the synergistic effects that occur among immune, proliferation, and metabolism modules. In conclusion, this approach deepens the understanding of the underlying mechanisms that cause inflammation to potentially increase the cancer risk of colonic epithelial cells and accelerate the translation into novel functional modules or synergistic module combinations that modulate complex disease phenotypes.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Koizumi Y, Fukushima J, Kobayashi Y, et al (2019)

Genome-Scale CRISPR/Cas9 Screening Reveals Squalene Epoxidase as a Susceptibility Factor for Cytotoxicity of Malformin A1.

Chembiochem : a European journal of chemical biology, 20(12):1563-1568.

Malformin A1 (MA1) is a fungus-produced cyclic pentapeptide. MA1 exhibits teratogenicity to plants, fibrinolysis-enhancing activity, and cytotoxicity to mammalian cells. To clarify the cytotoxic mechanism of MA1, we screened for the genes involved in the cytotoxicity of MA1 in monocytoid U937 cells by using a CRISPR/Cas9-based genome-wide knockout library. Screening was performed by positive selection for cells that were resistant to MA1 treatment, and single guide RNAs (sgRNAs) integrated into MA1-resistant cells were analyzed by high-throughput sequencing. As a result of the evaluation of sgRNAs that were enriched in MA1-resistant cells, SQLE, which encodes squalene epoxidase, was identified as a candidate gene. SQLE-depleted U937 cells were viable in the presence of MA1, and squalene epoxidase inhibitor conferred MA1 resistance to wild-type cells. These results indicate that squalene epoxidase is implicated in the cytotoxicity of MA1. This finding represents a new insight into applications of MA1 for treating ischemic diseases.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Geng P, Leonard SP, Mishler DM, et al (2019)

Synthetic Genome Defenses against Selfish DNA Elements Stabilize Engineered Bacteria against Evolutionary Failure.

ACS synthetic biology, 8(3):521-531.

Mobile genetic elements drive evolution by disrupting genes and rearranging genomes. Eukaryotes have evolved epigenetic mechanisms, including DNA methylation and RNA interference, that silence mobile elements and thereby preserve the integrity of their genomes. We created an artificial reprogrammable epigenetic system based on CRISPR interference to give engineered bacteria a similar line of defense against transposons and other selfish elements in their genomes. We demonstrate that this CRISPR interference against mobile elements (CRISPRi-ME) approach can be used to simultaneously repress two different transposon families in Escherichia coli, thereby increasing the evolutionary stability of costly protein expression. We further show that silencing a transposon in Acinetobacter baylyi ADP1 reduces mutation rates by a factor of 5, nearly as much as deleting all copies of this element from its genome. By deploying CRISPRi-ME on a broad-host-range vector, we have created a generalizable platform for stabilizing the genomes of engineered bacterial cells for applications in metabolic engineering and synthetic biology.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Mu W, Tang N, Cheng C, et al (2019)

In vitro transcribed sgRNA causes cell death by inducing interferon release.

Protein & cell, 10(6):461-465.

RevDate: 2020-04-08
CmpDate: 2020-04-06

White CW, Johnstone EKM, See HB, et al (2019)

NanoBRET ligand binding at a GPCR under endogenous promotion facilitated by CRISPR/Cas9 genome editing.

Cellular signalling, 54:27-34.

Bioluminescence resonance energy transfer (BRET) is a versatile tool used to investigate membrane receptor signalling and function. We have recently developed a homogenous NanoBRET ligand binding assay to monitor interactions between G protein-coupled receptors and fluorescent ligands. However, this assay requires the exogenous expression of a receptor fused to the nanoluciferase (Nluc) and is thus not applicable to natively-expressed receptors. To overcome this limitation in HEK293 cells, we have utilised CRISPR/Cas9 genome engineering to insert Nluc in-frame with the endogenous ADORA2B locus this resulted in HEK293 cells expressing adenosine A2B receptors under endogenous promotion tagged on their N-terminus with Nluc. As expected, we found relatively low levels of endogenous (gene-edited) Nluc/A2B receptor expression compared to cells transiently transfected with expression vectors coding for Nluc/A2B. However, in cells expressing gene-edited Nluc/A2B receptors we observed clear saturable ligand binding of a non-specific fluorescent adenosine receptor antagonist XAC-X-BY630 (Kd = 21.4 nM). Additionally, at gene-edited Nluc/A2B receptors we derived pharmacological parameters of ligand binding; Kd as well as Kon and Koff for binding of XAC-X-BY630 by NanoBRET association kinetic binding assays. Lastly, cells expressing gene-edited Nluc/A2B were used to determine the pKi of unlabelled adenosine receptor ligands in competition ligand binding assays. Utilising CRISPR/Cas9 genome engineering here we show that NanoBRET ligand binding assays can be performed at gene-edited receptors under endogenous promotion in live cells, therefore overcoming a fundamental limitation of NanoBRET ligand assays.

RevDate: 2020-04-08
CmpDate: 2020-04-07

Adkar SS, Wu CL, Willard VP, et al (2019)

Step-Wise Chondrogenesis of Human Induced Pluripotent Stem Cells and Purification Via a Reporter Allele Generated by CRISPR-Cas9 Genome Editing.

Stem cells (Dayton, Ohio), 37(1):65-76.

The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. Here, we demonstrate cartilaginous matrix production in three unique hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors (CPs) produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified CPs demonstrated an improved chondrogenic capacity compared with unselected populations. The ability to enrich for CPs and generate homogenous matrix without contaminating cell types will be essential for regenerative and disease modeling applications. Stem Cells 2019;37:65-76.

RevDate: 2020-04-08
CmpDate: 2020-04-06

An X, Li L, S Wu (2019)

In vivo tunable CRISPR mediates efficient somatic mutagenesis to generate tumor models.

Protein & cell, 10(6):450-454.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Callow MG, Watanabe C, Wickliffe KE, et al (2018)

CRISPR whole-genome screening identifies new necroptosis regulators and RIPK1 alternative splicing.

Cell death & disease, 9(3):261.

The necroptotic cell death pathway is a key component of human pathogen defense that can become aberrantly derepressed during tissue homeostasis to contribute to multiple types of tissue damage and disease. While formation of the necrosome kinase signaling complex containing RIPK1, RIPK3, and MLKL has been extensively characterized, additional mechanisms of its regulation and effector functions likely remain to be discovered. We screened 19,883 mouse protein-coding genes by CRISPR/Cas9-mediated gene knockout for resistance to cytokine-induced necroptosis and identified 112 regulators and mediators of necroptosis, including 59 new candidate pathway components with minimal or no effect on cell growth in the absence of necroptosis induction. Among these, we further characterized the function of PTBP1, an RNA binding protein whose activity is required to maintain RIPK1 protein abundance by regulating alternative splice-site selection.

RevDate: 2020-04-08
CmpDate: 2020-04-06

Cai S, Chen Y, Shang Y, et al (2018)

Knockout of zebrafish interleukin 7 receptor (IL7R) by the CRISPR/Cas9 system delays retinal neurodevelopment.

Cell death & disease, 9(3):273.

Interleukin 7 receptor (il7r), a transmembrane receptor, belongs to the type I cytokine receptor family. Il7r is involved in the pathogenesis of neurodegenerative disorders, such as multiple sclerosis. Targeted knockdown of il7r leads to delayed myelination, highlighting the potential role of il7r in the development of the nervous system. Zebrafish is an ideal model for the study of neurogenesis; moreover, the il7r gene is highly conserved between zebrafish and human. The aim of the present study was to investigate the novel function of il7r in neurogenesis. First, an il7r -/- homozygous mutant line was generated by clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (CRISPR/Cas9) technology. Second, the gross development of il7r-/- mutants revealed remarkably smaller eyes and delayed retinal neurodifferentiation. Third, microarray analysis revealed that genes associated with the phototransduction signalling pathway were strongly down-regulated in il7r -/- mutants. Finally, the results from behavioural tests indicated that visual function was impaired in il7r -/- mutant larvae. Overall, our data demonstrate that a lack of il7r retards the development of the retina. Thus, il7r is an essential molecule for maintaining normal retinal development in zebrafish.

RevDate: 2020-04-07

Xu M (2020)

CCR5-Δ32 biology, gene editing, and warnings for the future of CRISPR-Cas9 as a human and humane gene editing tool.

Cell & bioscience, 10:48 pii:410.

Background: Biomedical technologies have not just improved human health but also assisted in the creation of human life. Since the first birth of a healthy baby by in vitro fertilization (IVF) 40 years ago, IVF has been the mainstay treatment for couples struggling with infertility. This technology, in addition to increasingly accessible genetic testing, has made it possible for countless couples to have children. Since CRISPR-Cas9 gene editing was described in 2015, its potential for targeting genetic diseases has been much anticipated. However, the potential of using CRISPR-Cas9 for human germline modification has led to many fears of "designer babies" and widespread concerns for the impact of this technology on human evolution and its implications in Social Darwinism. In addition to these ethical/moral concerns, there remain many unknowns about CRISPR-Cas9 technology and endless unanticipated consequence to gene editing.

Methods: In this paper, we analyze the current progresses of CRISPR-Cas9 technology and discuss the theoretical advantages of certain allelic variances in the C-C chemokine receptor 5 gene (CCR5) in the setting of recent ethical/moral concerns regarding gene editing using the CRISPR-Cas9 system.

Results: These uncertainties have been highlighted recently by the birth of Chinese twins whose C-C chemokine receptor 5 (CCR5) gene had been inactivated via CRISPR-Cas9 to be theoretically protective against HIV infection. CCR5 signaling is critical for the successful infection of human immunodeficiency virus (HIV) and people with homozygous inactivating CCR5-Δ32 mutations have been shown to be protected against HIV infection. Those with the CCR5-Δ32/Δ32 mutation also have greater neuroplasticity, allowing for improved recovery from neurological trauma, and decreased Chagas cardiomyopathy. However, the CCR5-Δ32/Δ32 mutation has also been associated with earlier clinical manifestations for West Nile infection, ambiguous effects on osteoclast function, and a four-fold increased mortality from influenza infection. These detrimental health impacts, in addition to the confounding factor that these CRISPR babies do not carry this exact CCR5-Δ32/Δ32 mutation, lead to many questions regarding the children's future health and the moral conundrum of their birth. The creation and birth of these babies was not completed with any scientific, ethical, or governmental oversight, which has spurned the acceleration of talks regarding global regulations for human genetic editing.

Conclusions: Although we can try to regulate for ethical, health-related only use of this technology, moral and governmental oversights need to be supplemented by technical regulations. For instance, whole genome sequencing needs to be used to eliminate off-target mutations that could affect the health and safety of infants born to this process. Like Pandora's Box, we cannot pretend to forget CRISPR-Cas9 technology, all we can do is ensure a safe, moral, and equitable used of this technology.

RevDate: 2020-04-07

Álvarez VE, Quiroga MP, Galán AV, et al (2020)

Crucial Role of the Accessory Genome in the Evolutionary Trajectory of Acinetobacter baumannii Global Clone 1.

Frontiers in microbiology, 11:342.

Acinetobacter baumannii is one of the most important nosocomial pathogens able to rapidly develop extensive drug resistance. Here, we study the role of accessory genome in the success of the globally disseminated clone 1 (GC1) with functional and genomic approaches. Comparative genomics was performed with available GC1 genomes (n = 106) against other A. baumannii high-risk and sporadic clones. Genetic traits related to accessory genome were found common and conserved along time as two novel regions of genome plasticity, and a CRISPR-Cas system acquired before clonal diversification located at the same loci as "sedentary" modules. Although identified within hotspot for recombination, other block of accessory genome was also "sedentary" in lineage 1 of GC1 with signs of microevolution as the AbaR0-type genomic island (GI) identified in A144 and in A155 strains which were maintained one month in independent experiments without antimicrobial pressure. The prophage YMC/09/02/B1251_ABA_BP was found to be "mobile" since, although it was shared by all GC1 genomes, it showed high intrinsic microevolution as well as mobility to different insertion sites. Interestingly, a wide variety of Insertion Sequences (IS), probably acquired by the flow of plasmids related to Rep_3 superfamily was found. These IS showed dissimilar genomic location amongst GC1 genomes presumably associated with promptly niche adaptation. On the other hand, a type VI secretion system and three efflux pumps were subjected to deep processes of genomic loss in A. baumannii but not in GC1. As a whole, these findings suggest that preservation of some genetic modules of accessory genome harbored by strains from different continents in combination with great plasticity of IS and varied flow of plasmids, may be central features of the genomic structure of GC1. Competition of A144 and A155 versus A118 (ST 404/ND) without antimicrobial pressure suggested a higher ability of GC1 to grow over a clone with sporadic behavior which explains, from an ecological perspective, the global achievement of this successful pandemic clone in the hospital habitat. Together, these data suggest an essential role of still unknown properties of "mobile" and "sedentary" accessory genome that is preserved over time under different antibiotic or stress conditions.

RevDate: 2020-04-07

Rabinowitz R, Abadi S, Almog S, et al (2020)

Prediction of synonymous corrections by the BE-FF computational tool expands the targeting scope of base editing.

Nucleic acids research pii:5816857 [Epub ahead of print].

Base editing is a genome-editing approach that employs the CRISPR/Cas system to precisely install point mutations within the genome. A deaminase enzyme is fused to a deactivated Cas and enables transition conversions. The diversified repertoire of base editors provides a wide range of base editing possibilities. However, existing base editors cannot induce transversion substitutions and activate only within a specified region relative to the binding site, thus, they cannot precisely correct every point mutation. Here, we present BE-FF (Base Editors Functional Finder), a novel computational tool that identifies suitable base editors to correct the translated sequence erred by a point mutation. When a precise correction is impossible, BE-FF aims to mutate bystander nucleotides in order to induce synonymous corrections that will correct the coding sequence. To measure BE-FF practicality, we analysed a database of human pathogenic point mutations. Out of the transition mutations, 60.9% coding sequences could be corrected. Notably, 19.4% of the feasible corrections were not achieved by precise corrections but only by synonymous corrections. Moreover, 298 cases of transversion-derived pathogenic mutations were detected to be potentially repairable by base editing via synonymous corrections, although base editing is considered impractical for such mutations.

RevDate: 2020-04-07

Li F, Liu Q, Li X, et al (2020)

Construction of a new thermophilic fungus Myceliophthora thermophila platform for enzyme production using a versatile 2A peptide strategy combined with efficient CRISPR-Cas9 system.

Biotechnology letters pii:10.1007/s10529-020-02882-5 [Epub ahead of print].

OBJECTIVE: To construct a new thermophilic platform for glucoamylase production through 2A peptide strategy combined with CRISPR-Cas9 system using Myceliophthora thermophila as host, thermophilic filamentous fungus with industrial attractiveness to produce enzymes and chemicals from biomass.

RESULTS: We adapted the viral 2A peptide approach for M. thermophila and constructed a bicistronic vector for co-expressing two heterologous genes MhglaA and egfp. We obtained positive transformants OE-MhglaA-gfp overexpressing MhGlaA-9 ×His-2A-eGFP through convenient fluorescence screening, western blotting and RT-qPCR. We purified and characterized the recombinant MhGlaA, which exhibited stability in a broader pH range of 3.0-9.0 and thermostable stability at 65 °C, suggesting its potential industrial application. Furthermore, to improve glucoamylase secretion, we genetically engineered the obtained strain OE-MhglaA-gfp through our efficient CRISPR/Cas9 system and generated the quintuple mutant OE-MhglaA-gfpOE-amyRΔalp-1Δres-1Δcre-1, in which protein productivity and amylase activity were increased by approximately 12.0- and 8.2-fold compared with WT.

CONCLUSIONS: The 2A peptide approach worked well in M. thermophila and can be used to heterologously co-express two different proteins, and thus in combination with efficient CRISPR-Cas system will accelerate establishing hyper-secretion platforms for biotechnological applications.

RevDate: 2020-04-06

Cheng Q, Wei T, Farbiak L, et al (2020)

Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing.

Nature nanotechnology pii:10.1038/s41565-020-0669-6 [Epub ahead of print].

CRISPR-Cas gene editing and messenger RNA-based protein replacement therapy hold tremendous potential to effectively treat disease-causing mutations with diverse cellular origin. However, it is currently impossible to rationally design nanoparticles that selectively target specific tissues. Here, we report a strategy termed selective organ targeting (SORT) wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues via addition of a supplemental SORT molecule. Lung-, spleen- and liver-targeted SORT lipid nanoparticles were designed to selectively edit therapeutically relevant cell types including epithelial cells, endothelial cells, B cells, T cells and hepatocytes. SORT is compatible with multiple gene editing techniques, including mRNA, Cas9 mRNA/single guide RNA and Cas9 ribonucleoprotein complexes, and is envisioned to aid the development of protein replacement and gene correction therapeutics in targeted tissues.

RevDate: 2020-04-04

Meile S, Sarbach A, Du J, et al (2020)

Engineered reporter phages for rapid bioluminescence-based detection and differentiation of viable Listeria cells.

Applied and environmental microbiology pii:AEM.00442-20 [Epub ahead of print].

The pathogen Listeria monocytogenes causes listeriosis, a severe foodborne disease associated with high mortality. Rapid and sensitive methods are required for specific detection of this pathogen during food production. Bioluminescence-based reporter bacteriophages are genetically engineered viruses that infect their host cells with high specificity and transduce a heterologous luciferase gene, whose activity can be detected with high sensitivity to indicate the presence of viable target cells. Here, we use synthetic biology for de novo genome assembly and activation as well as CRISPR-Cas-assisted phage engineering to construct a set of reporter phages for the detection and differentiation of viable Listeria cells. Based on a single phage backbone, we compare the performance of four reporter phages that encode different crustacean, cnidarian and bacterial luciferases. From this panel of reporter proteins, nanoluciferase (NLuc) was identified as a superior enzyme and was subsequently introduced into the genomes of a broad host-range phage (A511) and two serovar 1/2- and serovar 4b/6a-specific Listeria phages (A006 and A500, respectively). The broad-range NLuc-based phage A511::nlucCPS detects one CFU of Listeria monocytogenes in 25 g of artificially contaminated milk, cold cut, and lettuce within less than 24 hours. In addition, this reporter phage successfully detected Listeria spp. in potentially contaminated natural food samples without producing false-positive or false-negative results. Finally, A006::nluc and A500::nluc enable serovar-specific Listeria diagnostics. In conclusion, these NLuc-based reporter phages enable rapid, ultrasensitive detection and differentiation of viable Listeria cells using a simple protocol that is 72 hours faster than culture-dependent approaches.Importance Culture-dependent methods are the gold standard for sensitive and specific detection of pathogenic bacteria within the food production chain. In contrast to molecular approaches, these methods detect viable cells, which is a key advantage for foods generated from heat-inactivated source material. However, culture-based diagnostics are typically much slower than molecular or proteomic strategies. Reporter phage assays combine the best of both worlds and allow for near on-line assessment of microbial safety because phage replication is extremely fast, highly target specific, and restricted to metabolically active host cells. In addition, reporter phage assays are inexpensive and do not require highly trained personnel, facilitating their on-site implementation. The reporter phages presented in this study not only allow for rapid detection, but also enable an early estimation of the potential virulence of Listeria isolates from food production and processing sites.

RevDate: 2020-04-03

Araldi RP, Khalil C, Grignet PH, et al (2020)

Medical Applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas) Tool: A Comprehensive Overview.

Gene pii:S0378-1119(20)30305-X [Epub ahead of print].

Since the discovery of the double helix and the introduction of genetic engineering, the possibility to develop new strategies to manipulate the genome has fascinated scientists around the world. Currently scientists have the knowledge andabilitytoedit the genomes. Several methodologies of gene editing have been established, all of them working like "scissor", creating double strand breaks at specific spots. The introduction of a new technology, which was adapted from the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas bacterial immune system, has revolutionized the genetic therapy field, as it allows a much more precise editing of gene than the previously described tools and, therefore, to prevent and treat disease in humans. This review aims to revisit the genome editing history that led to the rediscovery of the CRISPR/Cas technology and to explore the technical aspects, applications and perspectives of this fascinating, powerful, precise, simpler and cheaper technology in different fields.

RevDate: 2020-04-03

Mekler V, Kuznedelov K, K Severinov (2020)

Quantification of the affinities of CRISPR-Cas9 nucleases for cognate protospacer adjacent motif (PAM) sequences.

The Journal of biological chemistry pii:RA119.012239 [Epub ahead of print].

The CRISPR/Cas9 nucleases have been widely applied for genome editing in various organisms. Cas9 nucleases complexed with a guide RNA (Cas9/gRNA) find their targets by scanning and interrogating the genomic DNA for sequences complementary to the gRNA. Recognition of the DNA target sequence requires a short protospacer adjacent motif (PAM) located outside this sequence. Given that the efficiency of target location may depend on the strength of interactions that promote target recognition, here we sought to compare affinities of different Cas9 nucleases for their cognate PAM sequences. To this end, we measured affinities of Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, and Francisella novicida complexed with gRNAs (SpCas9/gRNA, SaCas9/gRNA, and FnCas9/gRNA, respectively) and of three engineered SpCas9/gRNA variants with altered PAM specificities for short, PAM-containing DNA probes. We used a "beacon" assay that measures the relative affinities of DNA probes by determining their ability to competitively affect the rate of Cas9/gRNA binding to fluorescently labeled target DNA derivatives called "Cas9 beacons." We observed significant differences in the affinities for cognate PAM sequences among the studied Cas9 enzymes. The relative affinities of SpCas9/gRNA and its engineered variants for canonical and suboptimal PAMs correlated with previous findings on the efficiency of these PAM sequences in genome editing. These findings suggest that high affinity of a Cas9 nuclease for its cognate PAM promotes higher genome-editing efficiency.

RevDate: 2020-04-03

Liu E, Zhang ZZ, Cheng X, et al (2020)

SCNrank: spectral clustering for network-based ranking to reveal potential drug targets and its application in pancreatic ductal adenocarcinoma.

BMC medical genomics, 13(Suppl 5):50 pii:10.1186/s12920-020-0681-6.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy. Due to its wide heterogeneity, PDAC acts aggressively and responds poorly to most chemotherapies, causing an urgent need for the development of new therapeutic strategies. Cell lines have been used as the foundation for drug development and disease modeling. CRISPR-Cas9 plays a key role in every step-in drug discovery: from target identification and validation to preclinical cancer cell testing. Using cell-line models and CRISPR-Cas9 technology together make drug target prediction feasible. However, there is still a large gap between predicted results and actionable targets in real tumors. Biological network models provide great modus to mimic genetic interactions in real biological systems, which can benefit gene perturbation studies and potential target identification for treating PDAC. Nevertheless, building a network model that takes cell-line data and CRISPR-Cas9 data as input to accurately predict potential targets that will respond well on real tissue remains unsolved.

METHODS: We developed a novel algorithm 'Spectral Clustering for Network-based target Ranking' (SCNrank) that systematically integrates three types of data: expression profiles from tumor tissue, normal tissue and cell-line PDAC; protein-protein interaction network (PPI); and CRISPR-Cas9 data to prioritize potential drug targets for PDAC. The whole algorithm can be classified into three steps: 1. using STRING PPI network skeleton, SCNrank constructs tissue-specific networks with PDAC tumor and normal pancreas tissues from expression profiles; 2. With the same network skeleton, SCNrank constructs cell-line-specific networks using the cell-line PDAC expression profiles and CRISPR-Cas 9 data from pancreatic cancer cell-lines; 3. SCNrank applies a novel spectral clustering approach to reduce data dimension and generate gene clusters that carry common features from both networks. Finally, SCNrank applies a scoring scheme called 'Target Influence score' (TI), which estimates a given target's influence towards the cluster it belongs to, for scoring and ranking each drug target.

RESULTS: We applied SCNrank to analyze 263 expression profiles, CRPSPR-Cas9 data from 22 different pancreatic cancer cell-lines and the STRING protein-protein interaction (PPI) network. With SCNrank, we successfully constructed an integrated tissue PDAC network and an integrated cell-line PDAC network, both of which contain 4414 selected genes that are overexpressed in tumor tissue samples. After clustering, 4414 genes are distributed into 198 clusters, which include 367 targets of FDA approved drugs. These drug targets are all scored and ranked by their TI scores, which we defined to measure their influence towards the network. We validated top-ranked targets in three aspects: Firstly, mapping them onto the existing clinical drug targets of PDAC to measure the concordance. Secondly, we performed enrichment analysis to these drug targets and the clusters there are within, to reveal functional associations between clusters and PDAC; Thirdly, we performed survival analysis for the top-ranked targets to connect targets with clinical outcomes. Survival analysis reveals that overexpression of three top-ranked genes, PGK1, HMMR and POLE2, significantly increases the risk of death in PDAC patients.

CONCLUSION: SCNrank is an unbiased algorithm that systematically integrates multiple types of omics data to do potential drug target selection and ranking. SCNrank shows great capability in predicting drug targets for PDAC. Pancreatic cancer-associated gene candidates predicted by our SCNrank approach have the potential to guide genetics-based anti-pancreatic drug discovery.

RevDate: 2020-04-03
CmpDate: 2020-04-03

Cheng Q, Dong L, Su T, et al (2019)

CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean.

BMC plant biology, 19(1):562.

BACKGROUND: Soybean (Glycine max) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants.

RESULTS: Here, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL (LHY) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using Agrobacterium rhizogenes strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of GmLHY displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in Gmlhy1a1b2a2b was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of GmLHY were significantly decreased in comparison to the WT. These results suggest that GmLHY encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies.

CONCLUSIONS: Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLHY genes reduces soybean plant height and shortens internodes from 20 to 35 days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.

RevDate: 2020-04-03
CmpDate: 2020-04-03

Cook NL, Pjanic M, Emmerich AG, et al (2019)

CRISPR-Cas9-mediated knockout of SPRY2 in human hepatocytes leads to increased glucose uptake and lipid droplet accumulation.

BMC endocrine disorders, 19(1):115.

BACKGROUND: The prevalence of obesity and its comorbidities, including type 2 diabetes mellitus (T2DM), is dramatically increasing throughout the world; however, the underlying aetiology is incompletely understood. Genome-wide association studies (GWAS) have identified hundreds of genec susceptibility loci for obesity and T2DM, although the causal genes and mechanisms are largely unknown. SPRY2 is a candidate gene identified in GWAS of body fat percentage and T2DM, and has recently been linked to insulin production in pancreatic β-cells. In the present study, we aimed to further understand SPRY2 via functional characterisation in HepG2 cells, an in vitro model of human hepatocytes widely used to investigate T2DM and insulin resistance.

METHODS: CRISPR-Cas9 genome editing was used to target SPRY2 in HepG2 cells, and the functional consequences of SPRY2 knockout (KO) and overexpression subsequently assessed using glucose uptake and lipid droplet assays, measurement of protein kinase phosphorylation and RNA sequencing.

RESULTS: The major functional consequence of SPRY2 KO was a significant increase in glucose uptake, along with elevated lipid droplet accumulation. These changes were attenuated, but not reversed, in cells overexpressing SPRY2. Phosphorylation of protein kinases across key signalling pathways (including Akt and mitogen activated protein kinases) was not altered after SPRY2 KO. Transcriptome profiling in SPRY2 KO and mock (control) cells revealed a number of differentially expressed genes related to cholesterol biosynthesis, cell cycle regulation and cellular signalling pathways. Phospholipase A2 group IIA (PLA2G2A) mRNA level was subsequently validated as significantly upregulated following SPRY2 KO, highlighting this as a potential mediator downstream of SPRY2.

CONCLUSION: These findings suggest a role for SPRY2 in glucose and lipid metabolism in hepatocytes and contribute to clarifying the function of this gene in the context of metabolic diseases.

RevDate: 2020-04-03
CmpDate: 2020-04-03

Guo P, Yang J, Huang J, et al (2019)

Therapeutic genome editing of triple-negative breast tumors using a noncationic and deformable nanolipogel.

Proceedings of the National Academy of Sciences of the United States of America, 116(37):18295-18303.

Triple-negative breast cancer (TNBC), which has the highest mortality rate of all breast cancer, is in urgent need of a therapeutic that hinders the spread and growth of cancer cells. CRISPR genome editing holds the promise of a potential cure for many genetic diseases, including TNBC; however, its clinical translation is being challenged by the lack of safe and effective nonviral delivery systems for in vivo therapeutic genome editing. Here we report the synthesis and application of a noncationic, deformable, and tumor-targeted nanolipogel system (tNLG) for CRISPR genome editing in TNBC tumors. We have demonstrated that tNLGs mediate a potent CRISPR knockout of Lipocalin 2 (Lcn2), a known breast cancer oncogene, in human TNBC cells in vitro and in vivo. The loss of Lcn2 significantly inhibits the migration and the mesenchymal phenotype of human TNBC cells and subsequently attenuates TNBC aggressiveness. In an orthotopic TNBC model, we have shown that systemically administered tNLGs mediated >81% CRISPR knockout of Lcn2 in TNBC tumor tissues, resulting in significant tumor growth suppression (>77%). Our proof-of-principle results provide experimental evidence that tNLGs can be used as a safe, precise, and effective delivery approach for in vivo CRISPR genome editing in TNBC.

RevDate: 2020-04-02

Lim S, Kim J, Kim Y, et al (2020)

CRISPR/Cas-directed programmable assembly of multi-enzyme complexes.

Chemical communications (Cambridge, England) [Epub ahead of print].

We describe a versatile CRISPR/Cas-based strategy to construct multi-enzyme complexes scaffolded on a DNA template in programmable patterns. Catalytically inactive dCas9 nuclease was used in combination with SpyCatcher-SpyTag chemistry to assemble enzymes in a highly modular fashion. Five enzymes comprising the violacein biosynthesis pathway were precisely organized in nanometer proximity; a notable increase in violacein production demonstrated the benefits of scaffolding.

RevDate: 2020-04-02

Fontana J, Dong C, Kiattisewee C, et al (2020)

Effective CRISPRa-mediated control of gene expression in bacteria must overcome strict target site requirements.

Nature communications, 11(1):1618 pii:10.1038/s41467-020-15454-y.

In bacterial systems, CRISPR-Cas transcriptional activation (CRISPRa) has the potential to dramatically expand our ability to regulate gene expression, but we lack predictive rules for designing effective gRNA target sites. Here, we identify multiple features of bacterial promoters that impose stringent requirements on CRISPRa target sites. Notably, we observe narrow, 2-4 base windows of effective sites with a periodicity corresponding to one helical turn of DNA, spanning ~40 bases and centered ~80 bases upstream of the TSS. However, we also identify two features suggesting the potential for broad scope: CRISPRa is effective at a broad range of σ70-family promoters, and an expanded PAM dCas9 allows the activation of promoters that cannot be activated by S. pyogenes dCas9. These results provide a roadmap for future engineering efforts to further expand and generalize the scope of bacterial CRISPRa.

RevDate: 2020-04-02

Vigouroux A, D Bikard (2020)

CRISPR Tools To Control Gene Expression in Bacteria.

Microbiology and molecular biology reviews : MMBR, 84(2): pii:84/2/e00077-19.

SUMMARYCRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Solomon SM (2020)

Genome editing in animals: why FDA regulation matters.

Nature biotechnology, 38(2):142-143.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Valderrama JA, Kulkarni SS, Nizet V, et al (2019)

A bacterial gene-drive system efficiently edits and inactivates a high copy number antibiotic resistance locus.

Nature communications, 10(1):5726.

Gene-drive systems in diploid organisms bias the inheritance of one allele over another. CRISPR-based gene-drive expresses a guide RNA (gRNA) into the genome at the site where the gRNA directs Cas9-mediated cleavage. In the presence of Cas9, the gRNA cassette and any linked cargo sequences are copied via homology-directed repair (HDR) onto the homologous chromosome. Here, we develop an analogous CRISPR-based gene-drive system for the bacterium Escherichia coli that efficiently copies a gRNA cassette and adjacent cargo flanked with sequences homologous to the targeted gRNA/Cas9 cleavage site. This "pro-active" genetic system (Pro-AG) functionally inactivates an antibiotic resistance marker on a high copy number plasmid with ~ 100-fold greater efficiency than control CRISPR-based methods, suggesting an amplifying positive feedback loop due to increasing gRNA dosage. Pro-AG can likewise effectively edit large plasmids or single-copy genomic targets or introduce functional genes, foreshadowing potential applications to biotechnology or biomedicine.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Uribe RV, van der Helm E, Misiakou MA, et al (2019)

Discovery and Characterization of Cas9 Inhibitors Disseminated across Seven Bacterial Phyla.

Cell host & microbe, 26(5):702.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Kumbha R, Hosny N, Matson A, et al (2020)

Efficient production of GGTA1 knockout porcine embryos using a modified handmade cloning (HMC) method.

Research in veterinary science, 128:59-68.

Handmade cloning is a zona-free nuclear transfer approach and an economical, efficient, and simple micromanipulation-free alternative to dolly based traditional cloning (TC). In this study, based on handmade cloning with minor modifications, an optimized bi-oocyte fusion (BOF) cloning method was established to produce GGTA1 KO porcine embryos using the CRISPR/Cas9 gene editing system. The GGTA1 gene is responsible for the generation of Gal epitopes on the surface of porcine cells, triggering hyperacute immune rejection in preclinical porcine-to-human xenotransplantation. The purpose of the present study is to establish an efficient protocol for activation of porcine oocyte cytoplast-fibroblast fused constructs developed to GGTA1 KO blastocysts by the zona-free bi-oocyte fusion cloning method. High percentages of cleavage (90 ± 2.6%) and blastocyst rates (39 ± 4.0%) were achieved upon treatment with demecolcine-assisted oocyte enucleation followed by 6 V alternating current for proper alignment and single-step fusion technique using a single direct current pulse of 1.0 kV/cm for 9 μs duration, compared to the double-step fusion method with combined chemical activation using thimerosal and dithiothreitol. Overall blastocyst rate was higher for oocyte enucleation by demecolcine (0.4 μg/ml) and 45 min incubation (42 ± 1.5%) compared to without demecolcine incubation followed by complete chemical thimerosal/dithiothreitol activation (33 ± 1.1%). The blastocyst rate (39 ± 1.0%) was found to be significantly higher 1 h post-electrofusion, compared to at 0 and 4 h (28 ± 1.5 and 6 ± 1.5%, respectively). Blastocyst development rates for GGTA1 knockout embryos (38 ± 1.76%) were comparable to those obtained with wild-type embryos (41.1 ± 0.67%). In conclusion, we achieved high overall efficiency in production of GGTA1 KO blastocysts by modified HMC protocol.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Lee JE, Neumann M, Duro DI, et al (2019)

CRISPR-based tools for targeted transcriptional and epigenetic regulation in plants.

PloS one, 14(9):e0222778.

Programmable gene regulators that can modulate the activity of selected targets in trans are a useful tool for probing and manipulating gene function. CRISPR technology provides a convenient method for gene targeting that can also be adapted for multiplexing and other modifications to enable strong regulation by a range of different effectors. We generated a vector toolbox for CRISPR/dCas9-based targeted gene regulation in plants, modified with the previously described MS2 system to amplify the strength of regulation, and using Golden Gate-based cloning to enable rapid vector assembly with a high degree of flexibility in the choice of promoters, effectors and targets. We tested the system using the floral regulator FLOWERING LOCUS T (FT) as a target and a range of different effector domains including the transcriptional activator VP64, the H3K27 acetyltransferase p300 and the H3K9 methyltransferase KRYPTONITE. When transformed into Arabidopsis thaliana, several of the constructs caused altered flowering time phenotypes that were associated with changes in FT expression and/or epigenetic status, thus demonstrating the effectiveness of the system. The MS2-CRISPR/dCas9 system can be used to modulate transcriptional activity and epigenetic status of specific target genes in plants, and provides a versatile tool that can easily be used with different targets and types of regulation for a range of applications.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Saito H, Y Yokobayashi (2019)

Editorial overview: Mammalian synthetic biology: from devices to multicellular systems.

Current opinion in chemical biology, 52:A1-A2.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Dok R, Bamps M, Glorieux M, et al (2020)

Radiosensitization approaches for HPV-positive and HPV-negative head and neck squamous carcinomas.

International journal of cancer, 146(4):1075-1085.

Radiotherapy is one of the most used treatment approaches for head and neck squamous cell carcinoma (HNSCC). Targeted inhibition of DNA repair machinery has the potential to improve treatment response by tailoring treatment to cancer cells lacking specific DNA repair pathways. Human papillomavirus (HPV)-negative and HPV-positive HNSCCs respond differently to radiotherapy treatment, suggesting that different approaches of DNA repair inhibition should be employed for these HNSCC groups. Here, we searched for optimal radiosensitization approaches for HPV-positive and HPV-negative HNSCCs by performing a targeted CRISPR-Cas9 screen. We found that inhibition of base excision repair resulted in a better radiotherapy response in HPV-positive HNSCC, which is correlated with upregulation of genes involved in base excision repair. In contrast, inhibition of nonhomologous end-joining and mismatch repair showed strong effects in both HNSCC groups. We validated the screen results by combining radiotherapy with targeted inhibition of DNA repair in several preclinical models including primary and recurrent patient-derived HNSCC xenografts. These findings underline the importance of stratifying HNSCC patients for combination treatments.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Hou L, Wang Y, Liu Y, et al (2019)

Paf1C regulates RNA polymerase II progression by modulating elongation rate.

Proceedings of the National Academy of Sciences of the United States of America, 116(29):14583-14592.

Elongation factor Paf1C regulates several stages of the RNA polymerase II (Pol II) transcription cycle, although it is unclear how it modulates Pol II distribution and progression in mammalian cells. We found that conditional ablation of Paf1 resulted in the accumulation of unphosphorylated and Ser5 phosphorylated Pol II around promoter-proximal regions and within the first 20 to 30 kb of gene bodies, respectively. Paf1 ablation did not impact the recruitment of other key elongation factors, namely, Spt5, Spt6, and the FACT complex, suggesting that Paf1 function may be mechanistically distinguishable from each of these factors. Moreover, loss of Paf1 triggered an increase in TSS-proximal nucleosome occupancy, which could impose a considerable barrier to Pol II elongation past TSS-proximal regions. Remarkably, accumulation of Ser5P in the first 20 to 30 kb coincided with reductions in histone H2B ubiquitylation within this region. Furthermore, we show that nascent RNA species accumulate within this window, suggesting a mechanism whereby Paf1 loss leads to aberrant, prematurely terminated transcripts and diminution of full-length transcripts. Importantly, we found that loss of Paf1 results in Pol II elongation rate defects with significant rate compression. Our findings suggest that Paf1C is critical for modulating Pol II elongation rates by functioning beyond the pause-release step as an "accelerator" over specific early gene body regions.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Ishiguro S, Mori H, N Yachie (2019)

DNA event recorders send past information of cells to the time of observation.

Current opinion in chemical biology, 52:54-62.

While current omics and single cell technologies have enabled measurements of high-resolution molecular snapshots of cells at a large scale, these technologies all require destruction of samples and prevent us from analyzing dynamic changes in molecular profiles, phenotypes, and behaviors of individual cells in a complex system. One possible direction to overcome this issue is the development of a cell-embedded 'event recorder' system, whereby molecular and phenotypic information of a cell(s) can be obtained at the time of observation with their past event information stored in 'heritable polymers' of the same cell. This concept has been demonstrated by many synthetic cellular circuits that monitor and transmit a certain set of environmental and intracellular signals into DNA, and have now been further accelerated by recent CRISPR-related technologies. Notably, the discovery of the RT-Cas1-Cas2 system, which acquires sequences of cellular transcripts into a specific host genomic region, has enabled recording of a broader range of molecular profile histories in the DNA tapes of cells, to understand the dynamics of complex biological processes that cannot be addressed by current technologies.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Jin UH, Karki K, Cheng Y, et al (2019)

The aryl hydrocarbon receptor is a tumor suppressor-like gene in glioblastoma.

The Journal of biological chemistry, 294(29):11342-11353.

The aryl hydrocarbon receptor (AhR) plays an important role in maintaining cellular homeostasis and also in pathophysiology. For example, the interplay between the gut microbiome and microbially derived AhR ligands protects against inflammation along the gut-brain axis. The AhR and its ligands also inhibit colon carcinogenesis, but it has been reported that the AhR and its ligand kynurenine enhance glioblastoma (GBM). In this study, using both established and patient-derived GBM cells, we re-examined the role of kynurenine and the AhR in GBM, observing that kynurenine does not modulate AhR-mediated gene expression and does not affect invasion of GBM cells. Therefore, using an array of approaches, including ChIP, quantitative real-time PCR, and cell migration assays, we primarily focused on investigating the role of the AhR in GBM at the functional molecular and genomic levels. The results of transient and stable CRISPR/Cas9-mediated AhR knockdown in GBM cells indicated that loss of AhR enhances GBM tumor growth in a mouse xenograft model, increases GBM cell invasion, and up-regulates expression of pro-invasion/pro-migration genes, as determined by ingenuity pathway analysis of RNA-Seq data. We conclude that the AhR is a tumor suppressor-like gene in GBM; future studies are required to investigate whether the AhR could be a potential drug target for treating patients with GBM who express this receptor.

RevDate: 2020-04-02
CmpDate: 2020-04-02

Lessard CB, Rodriguez E, Ladd TB, et al (2019)

Individual and combined presenilin 1 and 2 knockouts reveal that both have highly overlapping functions in HEK293T cells.

The Journal of biological chemistry, 294(29):11276-11285.

Presenilins 1 and 2 (PS1 and 2) are the catalytic subunits of γ-secretase, a multiprotein protease that cleaves amyloid protein precursor and other type I transmembrane proteins. Previous studies with mouse models or cells have indicated differences in PS1 and PS2 functions. We have recently reported that clinical γ-secretase inhibitors (GSIs), initially developed to manage Alzheimer's disease and now being considered for other therapeutic interventions, are both pharmacologically and functionally distinct. Here, using CRISPR/Cas9-based gene editing, we established human HEK 293T cell lines in which endogenous PS1, PS2, or both have been knocked out. Using these knockout lines to examine differences in PS1- and PS2-mediated cleavage events, we confirmed that PS2 generates more intracellular β-amyloid than does PS1. Moreover, we observed subtle differences in PS1- and PS2-mediated cleavages of select substrates. In exploring the question of whether differences in activity among clinical GSIs could be attributed to differential inhibition of PS1 or PS2, we noted that select GSIs inhibit PS1 and PS2 activities on specific substrates with slightly different potencies. We also found that endoproteolysis of select PS1 FAD-linked variants in human cells is more efficient than what has been previously reported for mouse cell lines. Overall, these results obtained with HEK293T cells suggest that selective PS1 or PS2 inhibition by a given GSI does not explain the previously observed differences in functional and pharmacological properties among various GSIs.

RevDate: 2020-04-02
CmpDate: 2020-04-02

McGuire CM, Collins MP, Sun-Wada G, et al (2019)

Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells.

The Journal of biological chemistry, 294(29):11248-11258.

The vacuolar H+-ATPase (V-ATPase) is an ATP-driven proton pump present in various intracellular membranes and at the plasma membrane of specialized cell types. Previous work has reported that plasma membrane V-ATPases are key players in breast cancer cell invasiveness. The two subunit a-isoforms known to target the V-ATPase to the plasma membrane are a3 and a4, and expression of a3 has been shown to correlate with plasma membrane localization of the V-ATPase in various invasive human breast cancer cell lines. Here we analyzed the role of subunit a-isoforms in the invasive mouse breast cancer cell line, 4T1-12B. Quantitation of mRNA levels for each isoform by quantitative RT-PCR revealed that a4 is the dominant isoform expressed in these cells. Using a CRISPR/Cas9-based approach to disrupt the genes encoding each of the four V-ATPase subunit a-isoforms, we found that ablation of only the a4-encoding gene significantly inhibits invasion and migration of 4T1-12B cells. Additionally, cells with disrupted a4 exhibited reduced V-ATPase expression at the leading edge, suggesting that the a4 isoform is primarily responsible for targeting the V-ATPase to the plasma membrane in 4T1-12B cells. These findings suggest that different subunit a-isoforms may direct V-ATPases to the plasma membrane of different invasive breast cancer cell lines. They further suggest that expression of V-ATPases at the cell surface is the primary factor that promotes an invasive cancer cell phenotype.

RevDate: 2020-04-02
CmpDate: 2020-04-02

An H, Skelt L, Notaro A, et al (2019)

ALS-linked FUS mutations confer loss and gain of function in the nucleus by promoting excessive formation of dysfunctional paraspeckles.

Acta neuropathologica communications, 7(1):7.

Mutations in the FUS gene cause amyotrophic lateral sclerosis (ALS-FUS). Mutant FUS is known to confer cytoplasmic gain of function but its effects in the nucleus are less understood. FUS is an essential component of paraspeckles, subnuclear bodies assembled on a lncRNA NEAT1. Paraspeckles may play a protective role specifically in degenerating spinal motor neurons. However it is still unknown how endogenous levels of mutant FUS would affect NEAT1/paraspeckles. Using novel cell lines with the FUS gene modified by CRISPR/Cas9 and human patient fibroblasts, we found that endogenous levels of mutant FUS cause accumulation of NEAT1 isoforms and paraspeckles. However, despite only mild cytoplasmic mislocalisation of FUS, paraspeckle integrity is compromised in these cells, as confirmed by reduced interaction of mutant FUS with core paraspeckle proteins NONO and SFPQ and increased NEAT1 extractability. This results in NEAT1 localisation outside paraspeckles, especially prominent under conditions of paraspeckle-inducing stress. Consistently, paraspeckle-dependent microRNA production, a readout for functionality of paraspeckles, is impaired in cells expressing mutant FUS. In line with the cellular data, we observed paraspeckle hyper-assembly in spinal neurons of ALS-FUS patients. Therefore, despite largely preserving its nuclear localisation, mutant FUS leads to loss (dysfunctional paraspeckles) and gain (excess of free NEAT1) of function in the nucleus. Perturbed fine structure and functionality of paraspeckles accompanied by accumulation of non-paraspeckle NEAT1 may contribute to the disease severity in ALS-FUS.

RevDate: 2020-04-01

Suárez GA, Dugan KR, Renda BA, et al (2020)

Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining.

Nucleic acids research pii:5813807 [Epub ahead of print].

One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1's native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.

RevDate: 2020-04-01

Calderón-Franco D, Lin Q, van Loosdrecht MCM, et al (2020)

Anticipating Xenogenic Pollution at the Source: Impact of Sterilizations on DNA Release From Microbial Cultures.

Frontiers in bioengineering and biotechnology, 8:171.

The dissemination of DNA and xenogenic elements across waterways is under scientific and public spotlight due to new gene-editing tools, such as do-it-yourself (DIY) CRISPR-Cas kits deployable at kitchen table. Over decades, prevention of spread of genetically modified organisms (GMOs), antimicrobial resistances (AMR), and pathogens from transgenic systems has focused on microbial inactivation. However, sterilization methods have not been assessed for DNA release and integrity. Here, we investigated the fate of intracellular DNA from cultures of model prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae) cells that are traditionally used as microbial chassis for genetic modifications. DNA release was tracked during exposure of these cultures to conventional sterilization methods. Autoclaving, disinfection with glutaraldehyde, and microwaving are used to inactivate broths, healthcare equipment, and GMOs produced at kitchen table. DNA fragmentation and PCR-ability were measured on top of cell viability and morphology. Impact of these methods on DNA integrity was verified on a template of free λ DNA. Intense regular autoclaving (121°C, 20 min) resulted in the most severe DNA degradation and lowest household gene amplification capacity: 1.28 ± 0.11, 2.08 ± 0.03, and 4.96 ± 0.28 logs differences to the non-treated controls were measured from E. coli, S. cerevisiae, and λ DNA, respectively. Microwaving exerted strong DNA fragmentation after 100 s of exposure when free λ DNA was in solution (3.23 ± 0.06 logs difference) but a minor effect was observed when DNA was released from E. coli and S. cerevisiae (0.24 ± 0.14 and 1.32 ± 0.02 logs differences with the control, respectively). Glutaraldehyde prevented DNA leakage by preserving cell structures, while DNA integrity was not altered. The results show that current sterilization methods are effective on microorganism inactivation but do not safeguard an aqueous residue exempt of biologically reusable xenogenic material, being regular autoclaving the most severe DNA-affecting method. Reappraisal of sterilization methods is required along with risk assessment on the emission of DNA fragments in urban systems and nature.

RevDate: 2020-04-01

Zhang Q, Zhang L, Ross P, et al (2020)

Comparative Genomics of Lactobacillus crispatus from the Gut and Vagina Reveals Genetic Diversity and Lifestyle Adaptation.

Genes, 11(4): pii:genes11040360.

Lactobacillus crispatus colonizes the human feces, human vagina, and the crops and ceca of chicken. To explore the genetic characteristics and evolutionary relationships of L. crispatus isolated from different niches, we selected 37 strains isolated from the human vagina (n = 17), human feces (n = 11), and chicken feces (n = 9), and used comparative genomics to explore the genetic information of L. crispatus from the feces and vagina. No significant difference was found in the three sources of genomic features such as genome size, GC content, and number of protein coding sequences (CDS). However, in a phylogenetic tree constructed based on core genes, vagina-derived L. crispatus and feces-derived strains were each clustered separately. Therefore, the niche exerted an important impact on the evolution of L. crispatus. According to gene annotation, the L. crispatus derived from the vagina possessed a high abundance of genes related to acid tolerance, redox reactions, pullulanase, and carbohydrate-binding modules (CBMs). These genes helped L. crispatus to better adapt to the acidic environment of the vagina and obtain more nutrients, maintaining its dominance in the vagina in competition with other strains. In feces-derived bacteria, more genes encoding CRISPR/Cas system, glycoside hydrolases (GHs) family, and tetracycline/lincomycin resistance genes were found to adapt to the complex intestinal environment. This study highlights the evolutionary relationship of L. crispatus strains isolated from the vagina and feces, and the adaptation of L. crispatus to the host environment.

RevDate: 2020-04-01
CmpDate: 2020-04-01

Komáromy AM (2020)

CRISPR-Cas9 Disruption of Aquaporin 1: An Alternative to Glaucoma Eye Drop Therapy?.

Molecular therapy : the journal of the American Society of Gene Therapy, 28(3):706-708.

RevDate: 2020-04-01
CmpDate: 2020-04-01

Ebright RY, Lee S, Wittner BS, et al (2020)

Deregulation of ribosomal protein expression and translation promotes breast cancer metastasis.

Science (New York, N.Y.), 367(6485):1468-1473.

Circulating tumor cells (CTCs) are shed into the bloodstream from primary tumors, but only a small subset of these cells generates metastases. We conducted an in vivo genome-wide CRISPR activation screen in CTCs from breast cancer patients to identify genes that promote distant metastasis in mice. Genes coding for ribosomal proteins and regulators of translation were enriched in this screen. Overexpression of RPL15, which encodes a component of the large ribosomal subunit, increased metastatic growth in multiple organs and selectively enhanced translation of other ribosomal proteins and cell cycle regulators. RNA sequencing of freshly isolated CTCs from breast cancer patients revealed a subset with strong ribosome and protein synthesis signatures; these CTCs expressed proliferation and epithelial markers and correlated with poor clinical outcome. Therapies targeting this aggressive subset of CTCs may merit exploration as potential suppressors of metastatic progression.

RevDate: 2020-04-01
CmpDate: 2020-04-01

Jang HK, S Bae (2020)

i-Silence, Please! An Alternative for Gene Disruption via Adenine Base Editors.

Molecular therapy : the journal of the American Society of Gene Therapy, 28(2):348-349.

RevDate: 2020-03-31

Pyenson NC, LA Marraffini (2020)

Co-evolution within structured bacterial communities results in multiple expansion of CRISPR loci and enhanced immunity.

eLife, 9: pii:53078.

Type II CRISPR-Cas systems provide immunity against phages and plasmids that infect bacteria through the insertion of a short sequence from the invader's genome, known as the 'spacer', into the CRISPR locus. Spacers are transcribed into guide RNAs that direct the Cas9 nuclease to its target on the invader. In liquid cultures, most bacteria acquire a single spacer. Multiple spacer integration is a rare event which significance for immunity is poorly understood. Here, we found that when phage infections occur on solid media, a high proportion of the surviving colonies display complex morphologies that contain cells with multiple spacers. This is the result of the viral-host co-evolution, in which the immunity provided by the initial acquired spacer is easily overcome by escaper phages. Our results reveal the versatility of CRISPR-Cas immunity, which can respond with both single or multiple spacer acquisition schemes to solve challenges presented by different environments.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Yamazaki H, Shirakawa K, Matsumoto T, et al (2020)

APOBEC3B reporter myeloma cell lines identify DNA damage response pathways leading to APOBEC3B expression.

PloS one, 15(1):e0223463.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) DNA cytosine deaminase 3B (A3B) is a DNA editing enzyme which induces genomic DNA mutations in multiple myeloma and in various other cancers. APOBEC family proteins are highly homologous so it is especially difficult to investigate the biology of specifically A3B in cancer cells. To easily and comprehensively investigate A3B function in myeloma cells, we used CRISPR/Cas9 to generate A3B reporter cells that contain 3×FLAG tag and IRES-EGFP sequences integrated at the end of the A3B gene. These reporter cells stably express 3xFLAG tagged A3B and the reporter EGFP and this expression is enhanced by known stimuli, such as PMA. Conversely, shRNA knockdown of A3B decreased EGFP fluorescence and 3xFLAG tagged A3B protein levels. We screened a series of anticancer treatments using these cell lines and identified that most conventional therapies, such as antimetabolites or radiation, exacerbated endogenous A3B expression, but recent molecular targeted therapeutics, including bortezomib, lenalidomide and elotuzumab, did not. Furthermore, chemical inhibition of ATM, ATR and DNA-PK suppressed EGFP expression upon treatment with antimetabolites. These results suggest that DNA damage triggers A3B expression through ATM, ATR and DNA-PK signaling.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Becú-Villalobos D (2019)

[CRISPR-CAS9 in medicine, the saga continues].

Medicina, 79(6):522-523.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Straiton J (2019)

Genetically modified humans: the X-Men of scientific research.

BioTechniques, 66(6):249-252.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Campenhout CV, Cabochette P, Veillard AC, et al (2019)

Guidelines for optimized gene knockout using CRISPR/Cas9.

BioTechniques, 66(6):295-302.

CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.

RevDate: 2020-03-31
CmpDate: 2020-03-31

Yang B, Yang L, J Chen (2019)

Development and Application of Base Editors.

The CRISPR journal, 2(2):91-104.

Base editing is emerging as a potent new strategy to achieve precise gene editing. By combining different nucleobase deaminases with Cas9 or Cpf1 proteins, several base editors have recently been developed to achieve targeted base conversions in different genomic contexts. Importantly, base editors have been successfully applied in animals, plants, and bacteria to induce precise substitutions at the single-base level with high efficiency. In this review, we summarize recent progress in the development and application of base editors and discuss some of the future directions of the technology.

RevDate: 2020-03-30
CmpDate: 2020-03-30

Borowicz P, Chan H, Medina D, et al (2020)

A simple and efficient workflow for generation of knock-in mutations in Jurkat T cells using CRISPR/Cas9.

Scandinavian journal of immunology, 91(4):e12862.

CRISPR/Cas9 is a powerful gene-editing tool allowing for specific gene manipulation at targeted sites in the genome. Here, we used CRISPR/Cas9-mediated gene editing to introduce single amino acid mutations into proteins involved in T cell receptor signalling pathways. Knock-in mutations were introduced in Jurkat T cells by homologous directed repair using single-stranded oligodeoxynucleotides. Specifically, we aimed to create targeted mutations at two loci within LCK, a constitutively expressed gene, and at three loci within SH2D2A, whose expression is induced upon T cell activation. Here, we present a simple workflow that can be applied by any laboratory equipped for cell culture work, utilizing basic flow cytometry, Western blotting and PCR techniques. Our data reveal that gene editing may be locus-dependent and can vary between target sites, also within a gene. In our two targeted genes, on average 2% of the clones harboured homozygous mutations as assessed by allele-specific PCR and subsequent sequencing. We highlight the importance of decreasing the clonal heterogeneity and developing robust screening methods to accurately select for correct knock-in mutations. Our workflow may be employed in other immune cell lines and acts as a useful approach for decoding functional mechanisms of proteins of interest.

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ESP Quick Facts

ESP Origins

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

ESP Support

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

ESP Rationale

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

ESP Goal

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

ESP Usage

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

ESP Content

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

ESP Help

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

ESP Plans

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

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CRISPR-Cas

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

Electronic Scholarly Publishing
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Papers in Classical Genetics

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

Digital Books

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

Timelines

ESP now offers a much improved and expanded collection of timelines, designed to give the user choice over subject matter and dates.

Biographies

Biographical information about many key scientists.

Selected Bibliographies

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

ESP Picks from Around the Web (updated 07 JUL 2018 )