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Bibliography on: CRISPR-Cas

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Robert J. Robbins is a biologist, an educator, a science administrator, a publisher, an information technologist, and an IT leader and manager who specializes in advancing biomedical knowledge and supporting education through the application of information technology. More About:  RJR | OUR TEAM | OUR SERVICES | THIS WEBSITE

RJR: Recommended Bibliography 24 Feb 2020 at 01:33 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: 2020-02-22

Hao M, Wang Z, Qiao H, et al (2020)

Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli.

Cells, 9(2): pii:cells9020467.

As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.

RevDate: 2020-02-21

Deem TL, Collins JB, DeVost MH, et al (2020)

Assessment of faithful interleukin-3 production by novel bicistronic interleukin-3 reporter mice.

Immunology letters pii:S0165-2478(19)30414-6 [Epub ahead of print].

Interleukin-3 (IL-3) is an important hematopoietic growth factor and immunregulatory cytokine. Although activated T helper cells represent a main source of IL-3, other cell types have been reported to express this cytokine. However, precise identification and quantification of the cells that produce IL-3 in vivo have not been performed. Therefore, we used a CRISPR/Cas approach to engineer mice containing a bicistronic mRNA linking a readily identifiable reporter, enhanced green fluorescent protein (ZsGreen1), to IL-3 expression. To characterize these novel reporter mice, we first examined ZsGreen1 expression by CD4 T cells subsets primed and activated in vitro. We found that activated Th1 cells expressed ∼4-fold higher levels of ZsGreen1 as compared to Th0 and Th2 cells. Endogenous IL-3 expression remained intact although reporter Th1 cells secreted ∼33% less IL-3 than similarly activated wild-type cells. To characterize the ability of reporter mice to accurately mark IL-3-producing cells in vivo, we infected mice with Nippostrongylus brasiliensis. Low but significant numbers of ZsGreen1+ CD4 T cells were detected in the mesenteric lymph nodes and lung following both primary and secondary infection. No difference in basophil and intestinal mast cell numbers were observed between infected reporter and wild-type mice indicating that reporter mice secreted IL-3 levels in vivo that results in IL-3-driven biological activities which are indistinguishable from those observed in corresponding wild-type mice. These IL-3 reporter mice will be a valuable resource to investigate IL-3-dependent immune responses in vivo.

RevDate: 2020-02-21

Frith KH (2020)

CRISPR-Cas: What Is It and Why Should Nurses Care?.

Nursing education perspectives, 41(2):136-137.

RevDate: 2020-02-18
CmpDate: 2020-02-18

Zhang S, Li X, Lin Q, et al (2019)

Synergizing CRISPR/Cas9 off-target predictions for ensemble insights and practical applications.

Bioinformatics (Oxford, England), 35(7):1108-1115.

MOTIVATION: The RNA-guided CRISPR/Cas9 system has been widely applied to genome editing. CRISPR/Cas9 system can effectively edit the on-target genes. Nonetheless, it has recently been demonstrated that many homologous off-target genomic sequences could be mutated, leading to unexpected gene-editing outcomes. Therefore, a plethora of tools were proposed for the prediction of off-target activities of CRISPR/Cas9. Nonetheless, each computational tool has its own advantages and drawbacks under diverse conditions. It is hardly believed that a single tool is optimal for all conditions. Hence, we would like to explore the ensemble learning potential on synergizing multiple tools with genomic annotations together to enhance its predictive abilities.

RESULTS: We proposed an ensemble learning framework which synergizes multiple tools together to predict the off-target activities of CRISPR/Cas9 in different combinations. Interestingly, the ensemble learning using AdaBoost outperformed other individual off-target predictive tools. We also investigated the effect of evolutionary conservation (PhyloP and PhastCons) and chromatin annotations (ChromHMM and Segway) and found that only PhyloP can enhance the predictive capabilities further. Case studies are conducted to reveal ensemble insights into the off-target predictions, demonstrating how the current study can be applied in different genomic contexts. The best prediction predicted by AdaBoost is up to 0.9383 (AUC) and 0.2998 (PRC) that outperforms other classifiers. This is ascribable to the fact that AdaBoost introduces a new weak classifier (i.e. decision stump) in each iteration to learn the DNA sequences that were misclassified as off-targets until a small error rate is reached iteratively.

The source codes are freely available on GitHub at https://github.com/Alexzsx/CRISPR.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RevDate: 2020-02-21
CmpDate: 2020-02-21

Mu W, Zhang Y, Xue X, et al (2019)

5' capped and 3' polyA-tailed sgRNAs enhance the efficiency of CRISPR-Cas9 system.

Protein & cell, 10(3):223-228.

RevDate: 2020-02-20

Aschenbrenner S, Kallenberger SM, Hoffmann MD, et al (2020)

Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity.

Science advances, 6(6):eaay0187 pii:aay0187.

The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.

RevDate: 2020-02-20

Kim S, Loeff L, Colombo S, et al (2020)

Selective loading and processing of prespacers for precise CRISPR adaptation.

Nature pii:10.1038/s41586-020-2018-1 [Epub ahead of print].

CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

RevDate: 2020-02-20

Bor B, Collins AJ, Murugkar PP, et al (2020)

Insights Obtained by Culturing Saccharibacteria With Their Bacterial Hosts.

Journal of dental research [Epub ahead of print].

Oral microbiome research has moved from asking "Who's there?" to "What are they doing?" Understanding what microbes "do" involves multiple approaches, including obtaining genomic information and examining the interspecies interactions. Recently we isolated a human oral Saccharibacteria (TM7) bacterium, HMT-952, strain TM7x, which is an ultrasmall parasite of the oral bacterium Actinomyces odontolyticus. The host-parasite interactions, such as phage-bacterium or Saccharibacteria-host bacterium, are understudied areas with large potential for insight. The Saccharibacteria phylum is a member of Candidate Phyla Radiation, a large lineage previously devoid of cultivated members. However, expanding our understanding of Saccharibacteria-host interactions requires examining multiple phylogenetically distinct Saccharibacteria-host pairs. Here we report the isolation of 3 additional Saccharibacteria species from the human oral cavity in binary coculture with their bacterial hosts. They were obtained by filtering ultrasmall Saccharibacteria cells free of other larger bacteria and inoculating them into cultures of potential host bacteria. The binary cocultures obtained could be stably passaged and studied. Complete closed genomes were obtained and allowed full genome analyses. All have small genomes (<1 Mb) characteristic of parasitic species and dramatically limited de novo synthetic pathway capabilities but include either restriction modification or CRISPR-Cas systems as part of an innate defense against foreign DNA. High levels of gene synteny exist among Saccharibacteria species. Having isolates growing in coculture with their hosts allowed time course studies of growth and parasite-host interactions by phase contrast, fluorescence in situ hybridization, and scanning electron microscopy. The cells of the 4 oral Saccharibacteria species are ultrasmall and could be seen attached to their larger Actinobacteria hosts. Parasite attachment appears to lead to host cell death and lysis. The successful cultivation of Saccharibacteria species has significantly expanded our understanding of these ultrasmall Candidate Phyla Radiation bacteria.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Jo A, Ringel-Scaia VM, McDaniel DK, et al (2020)

Fabrication and characterization of PLGA nanoparticles encapsulating large CRISPR-Cas9 plasmid.

Journal of nanobiotechnology, 18(1):16.

BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9 protein system is a revolutionary tool for gene therapy. Despite promising reports of the utility of CRISPR-Cas9 for in vivo gene editing, a principal problem in implementing this new process is delivery of high molecular weight DNA into cells.

RESULTS: Using poly(lactic-co-glycolic acid) (PLGA), a nanoparticle carrier was designed to deliver a model CRISPR-Cas9 plasmid into primary bone marrow derived macrophages. The engineered PLGA-based carriers were approximately 160 nm and fluorescently labeled by encapsulation of the fluorophore 6,13-bis(triisopropylsilylethynyl) pentacene (TIPS pentacene). An amine-end capped PLGA encapsulated 1.6 wt% DNA, with an encapsulation efficiency of 80%. Release studies revealed that most of the DNA was released within the first 24 h and corresponded to ~ 2-3 plasmid copies released per nanoparticle. In vitro experiments conducted with murine bone marrow derived macrophages demonstrated that after 24 h of treatment with the PLGA-encapsulated CRISPR plasmids, the majority of cells were positive for TIPS pentacene and the protein Cas9 was detectable within the cells.

CONCLUSIONS: In this work, plasmids for the CRISPR-Cas9 system were encapsulated in nanoparticles comprised of PLGA and were shown to induce expression of bacterial Cas9 in murine bone marrow derived macrophages in vitro. These results suggest that this nanoparticle-based plasmid delivery method can be effective for future in vivo applications of the CRISPR-Cas9 system.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Zhang WW, G Matlashewski (2019)

Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania.

mSphere, 4(4): pii:4/4/e00408-19.

CRISPR-Cas9 genome editing relies on an efficient double-strand DNA break (DSB) and repair. Contrary to mammalian cells, the protozoan parasite Leishmania lacks the most efficient nonhomologous end-joining pathway and uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair to repair DSBs. Here, we reveal that Leishmania predominantly uses single-strand annealing (SSA) (>90%) instead of MMEJ (<10%) for DSB repair (DSBR) following CRISPR targeting of the miltefosine transporter gene, resulting in 9-, 18-, 20-, and 29-kb sequence deletions and multiple gene codeletions. Strikingly, when targeting the Leishmania donovani LdBPK_241510 gene, SSA even occurred by using direct repeats 77 kb apart, resulting in the codeletion of 15 Leishmania genes, though with a reduced frequency. These data strongly indicate that DSBR is not efficient in Leishmania, which explains why more than half of DSBs led to cell death and why the CRISPR gene-targeting efficiency is low compared with that in other organisms. Since direct repeat sequences are widely distributed in the Leishmania genome, we predict that many DSBs created by CRISPR are repaired by SSA. It is also revealed that DNA polymerase theta is involved in both MMEJ and SSA in Leishmania Collectively, this study establishes that DSBR mechanisms and their competence in an organism play an important role in determining the outcome and efficacy of CRISPR gene targeting. These observations emphasize the use of donor DNA templates to improve gene editing specificity and efficiency in Leishmania In addition, we developed a novel Staphylococcus aureus Cas9 constitutive expression vector (pLdSaCN) for gene targeting in LeishmaniaIMPORTANCE Due to differences in double-strand DNA break (DSB) repair mechanisms, CRISPR-Cas9 gene editing efficiency can vary greatly in different organisms. In contrast to mammalian cells, the protozoan parasite Leishmania uses microhomology-mediated end joining (MMEJ) and, occasionally, homology-directed repair (HDR) to repair DSBs but lacks the nonhomologous end-joining pathway. Here, we show that Leishmania predominantly uses single-strand annealing (SSA) instead of MMEJ for DSB repairs (DSBR), resulting in large deletions that can include multiple genes. This strongly indicates that the overall DSBR in Leishmania is inefficient and therefore can influence the outcome of CRISPR-Cas9 gene editing, highlighting the importance of using a donor DNA to improve gene editing fidelity and efficiency in Leishmania.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Chen N, Hu Z, Yang Y, et al (2019)

Inactive Cas9 blocks vitreous-induced expression of Mdm2 and proliferation and survival of retinal pigment epithelial cells.

Experimental eye research, 186:107716.

Mouse double minute (MDM)2 single nucleotide polymorphism (SNP) 309G allele in the second promoter of MDM2 enhances vitreous-induced expression of Mdm2 and degradation of the tumor suppressor protein p53. This MDM2SNP309G contributes to certain cancer development and experimental proliferative vitreoretinopathy. The goal of this study is to discover a novel strategy to only block vitreous-induced expression of Mdm2 for preventing vitreous-induced cell proliferation and survival and thus find a potential novel strategy to treat proliferation-related diseases. We created two mutations (D10A and H840A) in Streptococcus pyogenes (Sp)Cas9 within the nuclease domains (RuvC1 and HNH, respectively) to render this SpCas9 nuclease dead named as dCas9 in a lentiCRISPR v2 vector. Then an MDM2-sgRNA targeting the second promoter of human MDM2 gene was cloned into this vector for producing lentivirus to infect human retinal pigment epithelial (RPE) cells with, which carry a heterozygous genotype of MDM2SNP309 T/G. lacZ-sgRNA was used as a control. As a result, we discovered that vitreous from experimental rabbits induced a 1.9 ± 0.2 fold increase in Mdm2 and a 2.0 ± 0.2 fold decrease in p53 in the RPE cells with dCas9/lacZ-sgRNA compared to those with dCas9/MDM2-sgRNA, suggesting that dCas9 under the guidance of the MDM2-sgRNA prevented RV-stimulated increase in Mdm2. In addition, we found that the rabbit vitreous significantly enhanced cell proliferation (1.5 ± 0.2 fold), survival against apoptosis (2.2 ± 0.2 fold), migration (10 ± 1.5%) and contraction (112.7 ± 14.1 mm2) of the cells with dCas9/lacZ-sgRNA compared with those with dCas9/MDM2-sgRNA. These results indicated that application of the dCas9 targeted to the P2 of MDM2 is a potential therapeutic approach to diseases due to the P2-driven aberrant expression of Mdm2 - such as proliferative vitreoretinopathy.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Anonymous (2019)

Clever chip designs for diagnostics.

Nature biomedical engineering, 3(6):417-418.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Bruch R, Urban GA, C Dincer (2019)

Unamplified gene sensing via Cas9 on graphene.

Nature biomedical engineering, 3(6):419-420.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Bae S, JS Kim (2018)

Machine learning finds Cas9-edited genotypes.

Nature biomedical engineering, 2(12):892-893.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Duan D (2018)

CRISPR alleviates muscular dystrophy in dogs.

Nature biomedical engineering, 2(11):795-796.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Hsu MN, YC Hu (2019)

Local magnetic activation of CRISPR.

Nature biomedical engineering, 3(2):83-84.

RevDate: 2020-02-20
CmpDate: 2020-02-20

Ortega-Escalante JA, Jasper R, SM Miller (2019)

CRISPR/Cas9 mutagenesis in Volvox carteri.

The Plant journal : for cell and molecular biology, 97(4):661-672.

Volvox carteri and other volvocine green algae comprise an excellent model for investigating developmental complexity and its origins. Here we describe a method for targeted mutagenesis in V. carteri using CRISPR/Cas9 components expressed from transgenes. We used V. carteri nitrate reductase gene (nitA) regulatory sequences to conditionally express Streptococcus pyogenes Cas9, and V. carteri U6 RNA gene regulatory sequences to constitutively express single-guide RNA (sgRNA) transcripts. Volvox carteri was bombarded with both Cas9 vector and one of several sgRNA vectors programmed to target different test genes (glsA, regA and invA), and transformants were selected for expression of a hygromycin-resistance marker present on the sgRNA vector. Hygromycin-resistant transformants grown with nitrate as sole nitrogen source (inducing for nitA) were tested for Cas9 and sgRNA expression, and for the ability to generate progeny with expected mutant phenotypes. Some transformants of a somatic regenerator (Reg) mutant strain receiving sgRNA plasmid with glsA protospacer sequence yielded progeny (at a rate of ~0.01%) with a gonidialess (Gls) phenotype similar to that observed for previously described glsA mutants, and sequencing of the glsA gene in independent mutants revealed short deletions within the targeted region of glsA, indicative of Cas9-directed non-homologous end joining. Similarly, bombardment of a morphologically wild-type strain with the Cas9 plasmid and sgRNA plasmids targeting regA or invA yielded regA and invA mutant transformants/progeny, respectively (at rates of 0.1-100%). The capacity to make precisely directed frameshift mutations should greatly accelerate the molecular genetic analysis of development in V. carteri, and of developmental novelty in the volvocine algae.

RevDate: 2020-02-19

Deng M, Liu Z, Chen B, et al (2020)

Locus-Specific Regulation of Xist Expression Using the CRISPR-Cas9-Based System.

DNA and cell biology [Epub ahead of print].

DNA methylation inhibitor or loss and gain of function of DNA methylation key players were widely used to investigate the regulation of X inactive-specific transcript (Xist) expression by DNA methylation, which results in global change of DNA methylation. Here, we reported a novel method for regulation of Xist using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. First, Xist expression was increased in 5-aza-2'-deoxycytidine-treated female goat fibroblast cells. Second, three single-guide RNAs (sgRNAs) that target the Xist differential methylation region (DMR) were inserted to deactivated Cas9 (dCas9) nuclease and the catalytic domain of the DNA methyltransferase Dnmt3a coexpression plasmid. Bisulfite PCR analysis and quantitative real-time PCR revealed that the methylation level of the DMR was significantly increased, while the expression of Xist was downregulated in all three sgRNAs, compared with the mock-transfected cells. Third, the methylation activity at the sites of 37 bp from the protospacer-adjacent motif sequence showed the strong change relative to the mock-transfected cells. Furthermore, genome-wide DNA methylation and expression of the DNA methylation key players were not statistically changed in all three sgRNAs. Therefore, we confirmed that Xist expression was regulated by DNA methylation, and directed DNA methylation of Xist DMR at locus-specific solution decreased Xist expression.

RevDate: 2020-02-19

Li J, Liu Y, Wang Y, et al (2020)

[Optimization of base editing in Corynebacterium glutamicum].

Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 36(1):143-151.

In recent years, CRISPR/Cas9-mediated base editing has been developed to a powerful genome editing tool, providing advantages such as without introducing double-stranded DNA break, a donor template and relying on host homologous recombination repair pathway, and has been widely applied in animals, plants, yeast and bacteria. In previous study, our group developed a multiplex automated base editing method (MACBETH) in the important industrial model strain Corynebacterium glutamicum. In this study, to further optimize the method and improve the base editing efficiency in C. glutamicum, we first constructed a green fluorescent protein (GFP) reporter-based detection system. The point mutation in the inactivated GFP protein can be edited to restore the GFP fluorescence. By combining with flow cytometry analysis, the base-editing efficiency can be quickly calculated. Then, the base editor with the target gRNA was constructed, and the editing efficiency with the initial editing condition was (13.11±0.21)%. Based on this result, the editing conditions were optimized and the result indicated that the best medium is CGXII, the best initial OD₆₀₀ of induction is 0.05, the best induction time is 20 h, and the best IPTG concentration is 0.01 mmol/L. After optimization, the editing efficiency was improved to (30.35±0.75)%, which was 1.3-fold of that in initial condition. Finally, endogenous genomic loci of C. glutamicum were selected to assess if the optimized condition can improve genome editing in other loci. Editing efficiency of different loci in optimized condition were improved to 1.7-2.5 fold of that in original condition, indicating the effectiveness and versatility of the optimized condition. Our research will promote the better application of base editing technology in C. glutamicum.

RevDate: 2020-02-19

Ouyang J, Xue S, Zhou Q, et al (2020)

[Research progress and applications of gene editing technology CRISPR/Cas in zebrafish].

Sheng wu gong cheng xue bao = Chinese journal of biotechnology, 36(1):1-12.

Clustered regularly interspaced short palindromic repeats (CRISPR) are acquired immune system in bacteria and archaea. This system is used in site-directed gene editing. Recently, scientists discovered new CRISPR-associated (Cas) proteins, in which Cas12a-mediated gene editing can significantly reduce the off-target rate. In this article, we review CRISPR/Cas system's discovery of history, composition, classification, and working principle. The latest research progress of the CRISPR/Cas system, and its application in zebrafish are introduced.

RevDate: 2020-02-19

Bradde S, Nourmohammad A, Goyal S, et al (2020)

The size of the immune repertoire of bacteria.

Proceedings of the National Academy of Sciences of the United States of America pii:1903666117 [Epub ahead of print].

Some bacteria and archaea possess an immune system, based on the CRISPR-Cas mechanism, that confers adaptive immunity against viruses. In such species, individual prokaryotes maintain cassettes of viral DNA elements called spacers as a memory of past infections. Typically, the cassettes contain several dozen expressed spacers. Given that bacteria can have very large genomes and since having more spacers should confer a better memory, it is puzzling that so little genetic space would be devoted by prokaryotes to their adaptive immune systems. Here, assuming that CRISPR functions as a long-term memory-based defense against a diverse landscape of viral species, we identify a fundamental tradeoff between the amount of immune memory and effectiveness of response to a given threat. This tradeoff implies an optimal size for the prokaryotic immune repertoire in the observational range.

RevDate: 2020-02-19

Brooks MR, Padilla-Vélez L, Khan TA, et al (2020)

Prophage-Mediated Disruption of Genetic Competence in Staphylococcus pseudintermedius.

mSystems, 5(1): pii:5/1/e00684-19.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is a major cause of soft tissue infections in dogs and occasionally infects humans. Hypervirulent multidrug-resistant (MDR) MRSP clones have emerged globally. The sequence types ST71 and ST68, the major epidemic clones of Europe and North America, respectively, have spread to other regions. The genetic factors underlying the success of these clones have not been investigated thoroughly. Here, we performed a comprehensive genomic analysis of 371 S. pseudintermedius isolates to dissect the differences between major clonal lineages. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, restriction-modification (RM), and CRISPR/Cas systems differs significantly among MRSP clones. The isolates with GyrA+GrlA mutations, conferring fluoroquinolone resistance, carry more of these genes than those without GyrA+GrlA mutations. ST71 and ST68 clones carry lineage-specific prophages with genes that are likely associated with their increased fitness and virulence. We have discovered that a prophage, SpST71A, is inserted within the comGA gene of the late competence operon comG in the ST71 lineage. A functional comG is essential for natural genetic competence, which is one of the major modes of horizontal gene transfer (HGT) in bacteria. The RM and CRISPR/Cas systems, both major genetic barriers to HGT, are also lineage specific. Clones harboring CRISPR/Cas or a prophage-disrupted comG exhibited less genetic diversity and lower rates of recombination than clones lacking these systems. After Listeria monocytogenes, this is the second example of prophage-mediated competence disruption reported in any bacteria. These findings are important for understanding the evolution and clonal expansion of MDR MRSP clones.IMPORTANCEStaphylococcus pseudintermedius is a bacterium responsible for clinically important infections in dogs and can infect humans. In this study, we performed genomic analysis of 371 S. pseudintermedius isolates to understand the evolution of antibiotic resistance and virulence in this organism. The analysis covered significant reported clones, including ST71 and ST68, the major epidemic clones of Europe and North America, respectively. We show that the prevalence of genes associated with antibiotic resistance, virulence, prophages, and horizontal gene transfer differs among clones. ST71 and ST68 carry prophages with novel virulence and antibiotic resistance genes. Importantly, site-specific integration of a prophage, SpST71A, has led to the disruption of the genetic competence operon comG in ST71 clone. A functional comG is essential for the natural uptake of foreign DNA and thus plays an important role in the evolution of bacteria. This study provides insight into the emergence and evolution of antibiotic resistance and virulence in S. pseudintermedius, which may help in efforts to combat this pathogen.

RevDate: 2020-02-18
CmpDate: 2020-02-18

Lutz S, Brion C, Kliebhan M, et al (2019)

DNA variants affecting the expression of numerous genes in trans have diverse mechanisms of action and evolutionary histories.

PLoS genetics, 15(11):e1008375.

DNA variants that alter gene expression contribute to variation in many phenotypic traits. In particular, trans-acting variants, which are often located on different chromosomes from the genes they affect, are an important source of heritable gene expression variation. However, our knowledge about the identity and mechanism of causal trans-acting variants remains limited. Here, we developed a fine-mapping strategy called CRISPR-Swap and dissected three expression quantitative trait locus (eQTL) hotspots known to alter the expression of numerous genes in trans in the yeast Saccharomyces cerevisiae. Causal variants were identified by engineering recombinant alleles and quantifying the effects of these alleles on the expression of a green fluorescent protein-tagged gene affected by the given locus in trans. We validated the effect of each variant on the expression of multiple genes by RNA-sequencing. The three variants differed in their molecular mechanism, the type of genes they reside in, and their distribution in natural populations. While a missense leucine-to-serine variant at position 63 in the transcription factor Oaf1 (L63S) was almost exclusively present in the reference laboratory strain, the two other variants were frequent among S. cerevisiae isolates. A causal missense variant in the glucose receptor Rgt2 (V539I) occurred at a poorly conserved amino acid residue and its effect was strongly dependent on the concentration of glucose in the culture medium. A noncoding variant in the conserved fatty acid regulated (FAR) element of the OLE1 promoter influenced the expression of the fatty acid desaturase Ole1 in cis and, by modulating the level of this essential enzyme, other genes in trans. The OAF1 and OLE1 variants showed a non-additive genetic interaction, and affected cellular lipid metabolism. These results demonstrate that the molecular basis of trans-regulatory variation is diverse, highlighting the challenges in predicting which natural genetic variants affect gene expression.

RevDate: 2020-02-18
CmpDate: 2020-02-18

Snedeker J, Gibbons WJ, Paulding DF, et al (2019)

Gpr63 is a modifier of microcephaly in Ttc21b mouse mutants.

PLoS genetics, 15(11):e1008467.

The primary cilium is a signaling center critical for proper embryonic development. Previous studies have demonstrated that mice lacking Ttc21b have impaired retrograde trafficking within the cilium and multiple organogenesis phenotypes, including microcephaly. Interestingly, the severity of the microcephaly in Ttc21baln/aln homozygous null mutants is considerably affected by the genetic background and mutants on an FVB/NJ (FVB) background develop a forebrain significantly smaller than mutants on a C57BL/6J (B6) background. We performed a Quantitative Trait Locus (QTL) analysis to identify potential genetic modifiers and identified two regions linked to differential forebrain size: modifier of alien QTL1 (Moaq1) on chromosome 4 at 27.8 Mb and Moaq2 on chromosome 6 at 93.6 Mb. These QTLs were validated by constructing congenic strains. Further analysis of Moaq1 identified an orphan G-protein coupled receptor (GPCR), Gpr63, as a candidate gene. We identified a SNP that is polymorphic between the FVB and B6 strains in Gpr63 and creates a missense mutation predicted to be deleterious in the FVB protein. We used CRISPR-Cas9 genome editing to create two lines of FVB congenic mice: one with the B6 sequence of Gpr63 and the other with a deletion allele leading to a truncation of the GPR63 C-terminal tail. We then demonstrated that Gpr63 can localize to the cilium in vitro. These alleles affect ciliary localization of GPR63 in vitro and genetically interact with Ttc21baln/aln as Gpr63;Ttc21b double mutants show unique phenotypes including spina bifida aperta and earlier embryonic lethality. This validated Gpr63 as a modifier of multiple Ttc21b neural phenotypes and strongly supports Gpr63 as a causal gene (i.e., a quantitative trait gene, QTG) within the Moaq1 QTL.

RevDate: 2020-02-19
CmpDate: 2020-02-19

Gorelik A, Bartual SG, Borodkin VS, et al (2019)

Genetic recoding to dissect the roles of site-specific protein O-GlcNAcylation.

Nature structural & molecular biology, 26(11):1071-1077.

Modification of specific Ser and Thr residues of nucleocytoplasmic proteins with O-GlcNAc, catalyzed by O-GlcNAc transferase (OGT), is an abundant posttranslational event essential for proper animal development and is dysregulated in various diseases. Due to the rapid concurrent removal by the single O-GlcNAcase (OGA), precise functional dissection of site-specific O-GlcNAc modification in vivo is currently not possible without affecting the entire O-GlcNAc proteome. Exploiting the fortuitous promiscuity of OGT, we show that S-GlcNAc is a hydrolytically stable and accurate structural mimic of O-GlcNAc that can be encoded in mammalian systems with CRISPR-Cas9 in an otherwise unperturbed O-GlcNAcome. Using this approach, we target an elusive Ser 405 O-GlcNAc site on OGA, showing that this site-specific modification affects OGA stability.

RevDate: 2020-02-18
CmpDate: 2020-02-18

Cram D, Kulkarni M, Buchwaldt M, et al (2019)

WheatCRISPR: a web-based guide RNA design tool for CRISPR/Cas9-mediated genome editing in wheat.

BMC plant biology, 19(1):474.

BACKGROUND: CRISPR/Cas9 gene editing has become a revolutionary technique for crop improvement as it can facilitate fast and efficient genetic changes without the retention of transgene components in the final plant line. Lack of robust bioinformatics tools to facilitate the design of highly specific functional guide RNAs (gRNAs) and prediction of off-target sites in wheat is currently an obstacle to effective application of CRISPR technology to wheat improvement.

DESCRIPTION: We have developed a web-based bioinformatics tool to design specific gRNAs for genome editing and transcriptional regulation of gene expression in wheat. A collaborative study between the Broad Institute and Microsoft Research used large-scale empirical evidence to devise algorithms (Doech et al., 2016, Nature Biotechnology 34, 184-191) for predicting the on-target activity and off-target potential of CRISPR/SpCas9 (Streptococcus pyogenes Cas9). We applied these prediction models to determine on-target specificity and potential off-target activity for individual gRNAs targeting specific loci in the wheat genome. The genome-wide gRNA mappings and the corresponding Doench scores predictive of the on-target and off-target activities were used to create a gRNA database which was used as a data source for the web application termed WheatCRISPR.

CONCLUSION: The WheatCRISPR tool allows researchers to browse all possible gRNAs targeting a gene or sequence of interest and select effective gRNAs based on their predicted high on-target and low off-target activity scores, as well as other characteristics such as position within the targeted gene. It is publicly available at https://crispr.bioinfo.nrc.ca/WheatCrispr/ .

RevDate: 2020-02-19
CmpDate: 2020-02-19

Sun W, Yang J, Cheng Z, et al (2019)

Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States.

Molecular cell, 76(6):938-952.e5.

High-resolution Cas9 structures have yet to reveal catalytic conformations due to HNH nuclease domain positioning away from the cleavage site. Nme1Cas9 and Nme2Cas9 are compact nucleases for in vivo genome editing. Here, we report structures of meningococcal Cas9 homologs in complex with sgRNA, dsDNA, or the AcrIIC3 anti-CRISPR protein. DNA-bound structures represent an early step of target recognition, a later HNH pre-catalytic state, the HNH catalytic state, and a cleaved-target-DNA-bound state. In the HNH catalytic state of Nme1Cas9, the active site is seen poised at the scissile phosphodiester linkage of the target strand, providing a high-resolution view of the active conformation. The HNH active conformation activates the RuvC domain. Our structures explain how Nme1Cas9 and Nme2Cas9 read distinct PAM sequences and how AcrIIC3 inhibits Nme1Cas9 activity. These structures provide insights into Cas9 domain rearrangements, guide-target engagement, cleavage mechanism, and anti-CRISPR inhibition, facilitating the optimization of these genome-editing platforms.

RevDate: 2020-02-19
CmpDate: 2020-02-19

Freije CA, Myhrvold C, Boehm CK, et al (2019)

Programmable Inhibition and Detection of RNA Viruses Using Cas13.

Molecular cell, 76(5):826-837.e11.

The CRISPR effector Cas13 could be an effective antiviral for single-stranded RNA (ssRNA) viruses because it programmably cleaves RNAs complementary to its CRISPR RNA (crRNA). Here, we computationally identify thousands of potential Cas13 crRNA target sites in hundreds of ssRNA viral species that can potentially infect humans. We experimentally demonstrate Cas13's potent activity against three distinct ssRNA viruses: lymphocytic choriomeningitis virus (LCMV); influenza A virus (IAV); and vesicular stomatitis virus (VSV). Combining this antiviral activity with Cas13-based diagnostics, we develop Cas13-assisted restriction of viral expression and readout (CARVER), an end-to-end platform that uses Cas13 to detect and destroy viral RNA. We further screen hundreds of crRNAs along the LCMV genome to evaluate how conservation and target RNA nucleotide content influence Cas13's antiviral activity. Our results demonstrate that Cas13 can be harnessed to target a wide range of ssRNA viruses and CARVER's potential broad utility for rapid diagnostic and antiviral drug development.

RevDate: 2020-02-19
CmpDate: 2020-02-19

Zhu X, Clarke R, Puppala AK, et al (2019)

Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.

Nature structural & molecular biology, 26(8):679-685.

The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg2+. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.

RevDate: 2020-02-19
CmpDate: 2020-02-19

Taylor DW (2019)

The final cut: Cas9 editing.

Nature structural & molecular biology, 26(8):669-670.

RevDate: 2020-02-19
CmpDate: 2020-02-19

Tracey WD (2019)

The taste of water.

eLife, 8: pii:48654.

Female mosquitos require a specific ion-channel protein to sense the presence of fresh water in which they can lay their eggs.

RevDate: 2020-02-18
CmpDate: 2020-02-18

Couch T, Murphy Z, Getman M, et al (2019)

Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.

Experimental hematology, 74:19-24.e4.

A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.

RevDate: 2020-02-19
CmpDate: 2020-02-19

Schmich F, Kuipers J, Merdes G, et al (2019)

netprioR: a probabilistic model for integrative hit prioritisation of genetic screens.

Statistical applications in genetics and molecular biology, 18(3): pii:/j/sagmb.ahead-of-print/sagmb-2018-0033/sagmb-2018-0033.xml.

In the post-genomic era of big data in biology, computational approaches to integrate multiple heterogeneous data sets become increasingly important. Despite the availability of large amounts of omics data, the prioritisation of genes relevant for a specific functional pathway based on genetic screening experiments, remains a challenging task. Here, we introduce netprioR, a probabilistic generative model for semi-supervised integrative prioritisation of hit genes. The model integrates multiple network data sets representing gene-gene similarities and prior knowledge about gene functions from the literature with gene-based covariates, such as phenotypes measured in genetic perturbation screens, for example, by RNA interference or CRISPR/Cas9. We evaluate netprioR on simulated data and show that the model outperforms current state-of-the-art methods in many scenarios and is on par otherwise. In an application to real biological data, we integrate 22 network data sets, 1784 prior knowledge class labels and 3840 RNA interference phenotypes in order to prioritise novel regulators of Notch signalling in Drosophila melanogaster. The biological relevance of our predictions is evaluated using in silico and in vivo experiments. An efficient implementation of netprioR is available as an R package at http://bioconductor.org/packages/netprioR.

RevDate: 2020-02-16

Lee J, Bayarsaikhan D, Bayarsaikhan G, et al (2020)

Recent advances in genome editing of stem cells for drug discovery and therapeutic application.

Pharmacology & therapeutics pii:S0163-7258(20)30029-2 [Epub ahead of print].

Genome engineering technologies right from viral vector-mediated to protein-based editing- which include zinc finger nucleases, TALENs, and CRISPR/Cas systems-have been improved significantly. These technologies have facilitated drug discovery and have resulted in the development of potential curative therapies for many intractable diseases. They can efficiently correct genetic errors; however, these technologies have limitations, such as off-target effects and possible safety issues, which need to be considered when employing these techniques in humans. Significant efforts have been made to overcome these limitations and to accelerate the clinical implementation of these technologies. In this review, we focus on the recent technological advancements in genome engineering and their applications in stem cells to enable efficient discovery of drugs and treatment of intractable diseases.

RevDate: 2020-02-17
CmpDate: 2020-02-17

Hampton T (2020)

DNA Prime Editing: A New CRISPR-Based Method to Correct Most Disease-Causing Mutations.

JAMA, 323(5):405-406.

RevDate: 2020-02-17
CmpDate: 2020-02-17

Narimani M, Sharifi M, Hakhamaneshi MS, et al (2019)

BIRC5 Gene Disruption via CRISPR/Cas9n Platform Suppress Acute Myelocytic Leukemia Progression.

Iranian biomedical journal, 23(6):369-378.

Background: Acute myelocytic leukemia (AML) is a clonal malignancy resulting from the accumulation of genetic abnormalities in the cells. Human baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5), encodes survivin, is one of only a handful of genes that is differentially over-expressed in numerous malignant diseases including AML.

Methods: The BIRC5 was silenced permanently in two AML cell lines, HL‑60 and KG-1, via the CRISPR/Cas9n system. After transfection of CRISPR constructs, genomic DNA was extracted and amplified to assess mutation detection. To evaluate BIRC5 gene expression, quantitative real-time PCR was performed. Also, MTT cell viability and Annexin‑V/propidium iodide flowcytometric staining were performed, and the data were analyzed using the Kolmogorov-Smirnov, Levene's, and ANOVA tests.

Results: The results indicated that Cas9n and its sgRNAs successfully triggered site-specific cleavage and mutation in the BIRC5 gene locus. Moreover, suppression of BIRC5 resulted in the reduction of cell viability, and induction of apoptosis and necrosis in HL60 and KG1 suggested that the permanent suppression of BIRC5 remarkably dropped the gene expression and cells viability.

Conclusion: This study reinforces the idea that BIRC5 disruption via Cas9n:sgRNAs has favorable effects on the AML clinical outcome. It thereby can be a promising candidate in a variety of leukemia treatments.

RevDate: 2020-02-17
CmpDate: 2020-02-17

Botella JR (2019)

Now for the hard ones: is there a limit on CRISPR genome editing in crops?.

Journal of experimental botany, 70(3):734-737.

RevDate: 2020-02-14

Wu M, Hu N, Du X, et al (2020)

Application of CRISPR/Cas9 technology in sepsis research.

Briefings in functional genomics pii:5732423 [Epub ahead of print].

CRISPR/Cas9, as a new genome-editing tool, offers new approaches to understand and treat diseases, which is being rapidly applied in various areas of biomedical research including sepsis field. The type II prokaryotic CRISPR/Cas system uses a single-guide RNA (sgRNA) to target the Cas9 nuclease to a specific genomic sequence, which is introduced into disease models for functional characterization and for testing of therapeutic strategies. This incredibly precise technology can be used for therapeutic research of gene-related diseases and to program any sequence in a target cell. Most importantly, the multifunctional capacity of this technology allows simultaneous editing of several genes. In this review, we focus on the basic principles, advantages and limitations of CRISPR/Cas9 and the use of the CRISPR/Cas9 system as a powerful tool in sepsis research and as a new strategy for the treatment of sepsis.

RevDate: 2020-02-14

He Q, Yu D, Bao M, et al (2020)

High-throughput and all-solution phase African Swine Fever Virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system.

Biosensors & bioelectronics, 154:112068 pii:S0956-5663(20)30065-8 [Epub ahead of print].

Here we report the development of a high throughput, all-solution phase, and isothermal detection system for African Swine Fever Virus (ASFV). CRISPR-Cas12a programmed with a CRISPR RNA (crRNA) is used to detect ASFV target DNA. Upon ASFV DNA binding, the Cas12a/crRNA/ASFV DNA complex becomes activated and degrades a fluorescent single stranded DNA (ssDNA) reporter present in the assay. We combine this powerful CRISPR-Cas assay with a fluorescence-based point-of-care (POC) system for rapid and accurate virus detection. Without nucleic acid amplification, a detection limit of 1 pM is achieved within 2 h. In addition, the ternary Cas12a/crRNA/ASFV DNA complex is highly stable at physiological temperature and continues to cleave the ssDNA reporter even after 24 h of incubation, resulting in an improved detection limit of 100 fM. We show that this system is very specific and can differentiate nucleic acid targets with closely matched sequences. The high sensitivity and selectivity of our system enables the detection of ASFV in femtomolar range. Importantly, this system features a disposable cartridge and a sensitive custom designed fluorometer, enabling compact and simple ASFV detection, intended for low resource settings.

RevDate: 2020-02-14

Hirotsune S, Kiyonari H, Jin M, et al (2020)

Enhanced homologous recombination by the modulation of targeting vector ends.

Scientific reports, 10(1):2518 pii:10.1038/s41598-020-58893-9.

The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.

RevDate: 2020-02-13

Rossi CC, Pereira MF, M Giambiagi-deMarval (2020)

Underrated Staphylococcus species and their role in antimicrobial resistance spreading.

Genetics and molecular biology, 43(1 suppl 2):e20190065 pii:S1415-47572020000300303.

The increasing threat of antimicrobial resistance has shed light on the interconnection between humans, animals, the environment, and their roles in the exchange and spreading of resistance genes. In this review, we present evidences that show that Staphylococcus species, usually referred to as harmless or opportunistic pathogens, represent a threat to human and animal health for acting as reservoirs of antimicrobial resistance genes. The capacity of genetic exchange between isolates of different sources and species of the Staphylococcus genus is discussed with emphasis on mobile genetic elements, the contribution of biofilm formation, and evidences obtained either experimentally or through genome analyses. We also discuss the involvement of CRISPR-Cas systems in the limitation of horizontal gene transfer and its suitability as a molecular clock to describe the history of genetic exchange between staphylococci.

RevDate: 2020-02-13

Chen X, Fan S, Wen C, et al (2020)

CRISPR/Cas9 for cancer treatment: technology, clinical applications and challenges.

Briefings in functional genomics pii:5734960 [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR) is described as RNA mediated adaptive immune system defense, which is naturally found in bacteria and archaea. CRISPR-Cas9 has shown great promise for cancer treatment in cancer immunotherapy, manipulation of cancer genome and epigenome and elimination or inactivation of carcinogenic viral infections. However, many challenges remain to be addressed to increase its efficacy, including off-target effects, editing efficiency, fitness of edited cells, immune response and delivery methods. Here, we explain CRISPR-Cas classification and its general function mechanism for gene editing. Then, we summarize these preclinical CRISPR-Cas9-based therapeutic strategies against cancer. Moreover, the challenges and improvements of CRISPR-Cas9 clinical applications will be discussed.

RevDate: 2020-02-13

Al-Shayeb B, Sachdeva R, Chen LX, et al (2020)

Clades of huge phages from across Earth's ecosystems.

Nature pii:10.1038/s41586-020-2007-4 [Epub ahead of print].

Bacteriophages typically have small genomes1 and depend on their bacterial hosts for replication2. Here we sequenced DNA from diverse ecosystems and found hundreds of phage genomes with lengths of more than 200 kilobases (kb), including a genome of 735 kb, which is-to our knowledge-the largest phage genome to be described to date. Thirty-five genomes were manually curated to completion (circular and no gaps). Expanded genetic repertoires include diverse and previously undescribed CRISPR-Cas systems, transfer RNAs (tRNAs), tRNA synthetases, tRNA-modification enzymes, translation-initiation and elongation factors, and ribosomal proteins. The CRISPR-Cas systems of phages have the capacity to silence host transcription factors and translational genes, potentially as part of a larger interaction network that intercepts translation to redirect biosynthesis to phage-encoded functions. In addition, some phages may repurpose bacterial CRISPR-Cas systems to eliminate competing phages. We phylogenetically define the major clades of huge phages from human and other animal microbiomes, as well as from oceans, lakes, sediments, soils and the built environment. We conclude that the large gene inventories of huge phages reflect a conserved biological strategy, and that the phages are distributed across a broad bacterial host range and across Earth's ecosystems.

RevDate: 2020-02-13

Spoto M, Guan C, Fleming E, et al (2020)

A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes.

mSphere, 5(1): pii:5/1/e00086-20.

The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful-focusing on CRISPRi as a potential application-as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species.IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

RevDate: 2020-02-13

Grigonyte AM, Harrison C, MacDonald PR, et al (2020)

Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7.

Viruses, 12(2): pii:v12020193.

With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.

RevDate: 2020-02-12

Burmistrz M, Krakowski K, A Krawczyk-Balska (2020)

RNA-Targeting CRISPR-Cas Systems and Their Applications.

International journal of molecular sciences, 21(3): pii:ijms21031122.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems have revolutionized modern molecular biology. Numerous types of these systems have been discovered to date. Many CRISPR-Cas systems have been used as a backbone for the development of potent research tools, with Cas9 being the most widespread. While most of the utilized systems are DNA-targeting, recently more and more attention is being gained by those that target RNA. Their ability to specifically recognize a given RNA sequence in an easily programmable way makes them ideal candidates for developing new research tools. In this review we summarize current knowledge on CRISPR-Cas systems which have been shown to target RNA molecules, that is type III (Csm/Cmr), type VI (Cas13), and type II (Cas9). We also present a list of available technologies based on these systems.

RevDate: 2020-02-12

Aquino-Jarquin G (2020)

Novel Engineered Programmable Systems for ADAR-Mediated RNA Editing.

Molecular therapy. Nucleic acids, 19:1065-1072 pii:S2162-2531(20)30026-3 [Epub ahead of print].

One of the most prevalent forms of post-transcriptional RNA modification is the conversion of adenosine-to-inosine (A-to-I), mediated by adenosine deaminase acting on RNA (ADAR) enzymes. The advent of the CRISPR/Cas systems inspires researchers to work actively in the engineering of programmable RNA-guided machines for basic research and biomedical applications. In this regard, CIRTS (CRISPR-Cas-Inspired RNA Targeting System), RESCUE (RNA Editing for Specific C to U Exchange), RESTORE (Recruiting Endogenous ADAR to Specific Transcripts for Oligonucleotide-mediated RNA Editing), and LEAPER (Leveraging Endogenous ADAR for Programmable Editing of RNA) are innovative RNA base-editing platforms that have recently been engineered to perform programmable base conversions on target RNAs mediated by ADAR enzymes in mammalian cells. Thus, these four currently characterized RNA-editing systems constitute novel molecular tools with compelling programmability, specificity, and efficiency that show us some creative ways to take advantage of the engineered deaminases for precise base editing. Moreover, the advanced engineering of these systems permits editing of full-length transcripts containing disease-causing point mutations without the loss of genomic information, providing an attractive alternative for in vivo research and in the therapeutic setting if the challenges encountered in off-target edits and delivery are appropriately addressed. Here, I present an analytical approach of the current status and rapid progress of the novel ADAR-mediated RNA-editing systems when highlighting the qualities of each new RNA-editing platform and how these RNA-targeting strategies could be used to recruit human ADARs on endogenous transcripts, not only for our understanding of RNA-modification-mediated regulation of gene expression but also for editing clinically relevant mutations in a programmable and straightforward manner.

RevDate: 2020-02-14

Wimmer F, CL Beisel (2019)

CRISPR-Cas Systems and the Paradox of Self-Targeting Spacers.

Frontiers in microbiology, 10:3078.

CRISPR-Cas immune systems in bacteria and archaea record prior infections as spacers within each system's CRISPR arrays. Spacers are normally derived from invasive genetic material and direct the immune system to complementary targets as part of future infections. However, not all spacers appear to be derived from foreign genetic material and instead can originate from the host genome. Their presence poses a paradox, as self-targeting spacers would be expected to induce an autoimmune response and cell death. In this review, we discuss the known frequency of self-targeting spacers in natural CRISPR-Cas systems, how these spacers can be incorporated into CRISPR arrays, and how the host can evade lethal attack. We also discuss how self-targeting spacers can become the basis for alternative functions performed by CRISPR-Cas systems that extend beyond adaptive immunity. Overall, the acquisition of genome-targeting spacers poses a substantial risk but can aid in the host's evolution and potentially lead to or support new functionalities.

RevDate: 2020-02-10

Wang J, Zhang C, B Feng (2020)

The rapidly advancing Class 2 CRISPR-Cas technologies: A customizable toolbox for molecular manipulations.

Journal of cellular and molecular medicine [Epub ahead of print].

The CRISPR-Cas technologies derived from bacterial and archaeal adaptive immune systems have emerged as a series of groundbreaking nucleic acid-guided gene editing tools, ultimately standing out among several engineered nucleases because of their high efficiency, sequence-specific targeting, ease of programming and versatility. Facilitated by the advancement across multiple disciplines such as bioinformatics, structural biology and high-throughput sequencing, the discoveries and engineering of various innovative CRISPR-Cas systems are rapidly expanding the CRISPR toolbox. This is revolutionizing not only genome editing but also various other types of nucleic acid-guided manipulations such as transcriptional control and genomic imaging. Meanwhile, the adaptation of various CRISPR strategies in multiple settings has realized numerous previously non-existing applications, ranging from the introduction of sophisticated approaches in basic research to impactful agricultural and therapeutic applications. Here, we summarize the recent advances of CRISPR technologies and strategies, as well as their impactful applications.

RevDate: 2020-02-11

Pan M, R Barrangou (2020)

Combining omics technologies with CRISPR-based genome editing to study food microbes.

Current opinion in biotechnology, 61:198-208 pii:S0958-1669(19)30165-X [Epub ahead of print].

The implementation of omics technologies such as genomics, proteomics and transcriptomics has revolutionized our understanding of microbiomes, and shed light on the functional attributes and mechanisms of action underlying the ability of probiotics to impact host health and starter cultures to drive food fermentation. Recently, molecular machines from CRISPR-Cas systems have redefined the gene editing toolbox and democritized our ability to alter the genome of food microorganisms. An integrated approach in which CRISPR-based genome editing is informed by omics studies is poised to enable the engineering of microorganisms and the formulation of microbiomes impacting the food supply chain. Here, we highlight the current applications of omics technologies in food microorganisms and CRISPR-based genome editing technologies in bacteria, and discuss how this integrated approach enables effective engineering of food microbes to generate enhanced probiotic strains, develop novel biotherapeutics and alter microbial communities in food matrices.

RevDate: 2020-02-07

Yuan C, Tian T, Sun J, et al (2020)

Universal and Naked-Eye Gene Detection Platform Based on CRISPR/Cas12a/13a System.

Analytical chemistry [Epub ahead of print].

Gold nanoparticles-based colorimetric assay is an attractive detection format, but is limited by the tedious and ineffective post-hybridization manipulations for genomic analysis. Here, we present a novel design for a colorimetric gene-sensing platform based on the CRISPR/Cas system. In this strategy, programmable recognition of DNA by Cas12a/crRNA and RNA by Cas13a/crRNA with a complementary target activates the trans-ssDNA or -ssRNA cleavage. Target-induced trans-ssDNA or ssRNA cleavage triggers an aggregation behavior change for the designed AuNPs-DNA probes pair, enabling the completion of naked-eye gene detection (transgenic rice, African swine fever virus, and miRNAs as the models) within 1 hour. This platform is also showing promise as a fast and inexpensive tool for bacteria identification using 16S ribosomal DNA or 16S ribosomal RNA. CRISPR/Cas-based colorimetric platform shows superior characteristics, such as probe universality, compatibility with isothermal reaction conditions, on-site detection capability, and high sensitivity; thus represents a robust next-generation gene detection platform.

RevDate: 2020-02-06

Kempton HR, Goudy LE, Love KS, et al (2020)

Multiple Input Sensing and Signal Integration Using a Split Cas12a System.

Molecular cell pii:S1097-2765(20)30037-X [Epub ahead of print].

The ability to integrate biological signals and execute a functional response when appropriate is critical for sophisticated cell engineering using synthetic biology. Although the CRISPR-Cas system has been harnessed for synthetic manipulation of the genome, it has not been fully utilized for complex environmental signal sensing, integration, and actuation. Here, we develop a split dCas12a platform and show that it allows for the construction of multi-input, multi-output logic circuits in mammalian cells. The system is highly programmable and can generate expandable AND gates with two, three, and four inputs. It can also incorporate NOT logic by using anti-CRISPR proteins as an OFF switch. By coupling the split dCas12a design to multiple tumor-relevant promoters, we provide a proof of concept that the system can implement logic gating to specifically detect breast cancer cells and execute therapeutic immunomodulatory responses.

RevDate: 2020-02-06

Holmes AC, Zagnoli-Vieira G, Caldecott KW, et al (2020)

Effects of TDP2/VPg Unlinkase Activity on Picornavirus Infections Downstream of Virus Translation.

Viruses, 12(2): pii:v12020166.

In this study, we characterized the role of host cell protein tyrosyl-DNA phosphodiesterase 2 (TDP2) activity, also known as VPg unlinkase, in picornavirus infections in a human cell model of infection. TDP2/VPg unlinkase is used by picornaviruses to remove the small polypeptide, VPg (Virus Protein genome-linked, the primer for viral RNA synthesis), from virus genomic RNA. We utilized a CRISPR/Cas-9-generated TDP2 knock out (KO) human retinal pigment epithelial-1 (hRPE-1) cell line, in addition to the wild type (WT) counterpart for our studies. We determined that in the absence of TDP2, virus growth kinetics for two enteroviruses (poliovirus and coxsackievirus B3) were delayed by about 2 h. Virus titers were reduced by ~2 log10 units for poliovirus and 0.5 log10 units for coxsackievirus at 4 hours post-infection (hpi), and by ~1 log10 unit at 6 hpi for poliovirus. However, virus titers were nearly indistinguishable from those of control cells by the end of the infectious cycle. We determined that this was not the result of an alternative source of VPg unlinkase activity being activated in the absence of TPD2 at late times of infection. Viral protein production in TDP2 KO cells was also substantially reduced at 4 hpi for poliovirus infection, consistent with the observed growth kinetics delay, but reached normal levels by 6 hpi. Interestingly, this result differs somewhat from what has been reported previously for the TDP2 KO mouse cell model, suggesting that either cell type or species-specific differences might be playing a role in the observed phenotype. We also determined that catalytically inactive TDP2 does not rescue the growth defect, confirming that TDP2 5' phosphodiesterase activity is required for efficient virus replication. Importantly, we show for the first time that polysomes can assemble efficiently on VPg-linked RNA after the initial round of translation in a cell culture model, but both positive and negative strand RNA production is impaired in the absence of TDP2 at mid-times of infection, indicating that the presence of VPg on the viral RNA affects a step in the replication cycle downstream of translation (e.g., RNA synthesis). In agreement with this conclusion, we found that double-stranded RNA production (a marker of viral RNA synthesis) is delayed in TDP2 KO RPE-1 cells. Moreover, we show that premature encapsidation of nascent, VPg-linked RNA is not responsible for the observed virus growth defect. Our studies provide the first lines of evidence to suggest that either negative- or positive-strand RNA synthesis (or both) is a likely candidate for the step that requires the removal of VPg from the RNA for an enterovirus infection to proceed efficiently.

RevDate: 2020-02-04

Yue JJ, Hong CY, Wei P, et al (2020)

How to start your monocot CRISPR/Cas project: plasmid design, efficiency detection, and offspring analysis.

Rice (New York, N.Y.), 13(1):9 pii:10.1186/s12284-019-0354-2.

The breakthrough CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9-mediated genome-editing technology has led to great progress in monocot research; however, several factors need to be considered for the efficient implementation of this technology. To generate genome-edited crops, single guide (sg)RNA and Cas9 DNA are delivered into plant cells and expressed, and the predicted position is targeted. Analyses of successful targeted mutations have revealed that the expression levels, expression timing, and variants of both sgRNA and Cas9 need to be sophisticatedly regulated; therefore, the promoters of these genes and the target site positions are the key factors for genome-editing efficiency. Currently, various vectors and online tools are available to aid sgRNA design. Furthermore, to reduce the sequence limitation of the protospacer adjacent motif (PAM) and for other purposes, many Cas protein variants and base editors can be used in plants. Before the stable transformation of a plant, the evaluation of vectors and target sites is therefore very important. Moreover, the delivery of Cas9-sgRNA ribonucleoproteins (RNPs) is one strategy that can be used to prevent transgene issues with the expression of sgRNA and Cas proteins. RNPs can be used to efficiently generate transgene-free genome-edited crops that can reduce transgene issues related to the generation of genetically modified organisms. In this review, we introduce new techniques for genome editing and identifying marker-free genome-edited mutants in monocot crops. Four topics are covered: the design and construction of plasmids for genome editing in monocots; alternatives to SpCas9; protoplasts and CRISPR; and screening for marker-free CRISPR/Cas9-induced mutants. We have aimed to encompass a full spectrum of information for genome editing in monocot crops.

RevDate: 2020-02-07

Smargon AA, Shi YJ, GW Yeo (2020)

RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering.

Nature cell biology, 22(2):143-150.

Deployment of RNA-guided DNA endonuclease CRISPR-Cas technology has led to radical advances in biology. As the functional diversity of CRISPR-Cas and parallel systems is further explored, RNA manipulation is emerging as a powerful mode of CRISPR-based engineering. In this Perspective, we chart progress in the RNA-targeting CRISPR-Cas (RCas) field and illustrate how continuing evolution in scientific discovery translates into applications for RNA biology and insights into mysteries, obstacles, and alternative technologies that lie ahead.

RevDate: 2020-02-06

Zhou X, Yang B, Stanton C, et al (2020)

Comparative analysis of Lactobacillus gasseri from Chinese subjects reveals a new species-level taxa.

BMC genomics, 21(1):119.

BACKGROUND: Lactobacillus gasseri as a probiotic has history of safe consumption is prevalent in infants and adults gut microbiota to maintain gut homeostasis.

RESULTS: In this study, to explore the genomic diversity and mine potential probiotic characteristics of L. gasseri, 92 strains of L. gasseri were isolated from Chinese human feces and identified based on 16 s rDNA sequencing, after draft genomes sequencing, further average nucleotide identity (ANI) value and phylogenetic analysis reclassified them as L. paragasseri (n = 79) and L. gasseri (n = 13), respectively. Their pan/core-genomes were determined, revealing that L. paragasseri had an open pan-genome. Comparative analysis was carried out to identify genetic features, and the results indicated that 39 strains of L. paragasseri harboured Type II-A CRISPR-Cas system while 12 strains of L. gasseri contained Type I-E and II-A CRISPR-Cas systems. Bacteriocin operons and the number of carbohydrate-active enzymes were significantly different between the two species.

CONCLUSIONS: This is the first time to study pan/core-genome of L. gasseri and L. paragasseri, and compare their genetic diversity, and all the results provided better understating on genetics of the two species.

RevDate: 2020-02-03

Shiriaeva A, Fedorov I, Vyhovskyi D, et al (2020)

Detection of CRISPR adaptation.

Biochemical Society transactions pii:222017 [Epub ahead of print].

Prokaryotic adaptive immunity is built when short DNA fragments called spacers are acquired into CRISPR (clustered regularly interspaced short palindromic repeats) arrays. CRISPR adaptation is a multistep process which comprises selection, generation, and incorporation of prespacers into arrays. Once adapted, spacers provide immunity through the recognition of complementary nucleic acid sequences, channeling them for destruction. To prevent deleterious autoimmunity, CRISPR adaptation must therefore be a highly regulated and infrequent process, at least in the absence of genetic invaders. Over the years, ingenious methods to study CRISPR adaptation have been developed. In this paper, we discuss and compare methods that detect CRISPR adaptation and its intermediates in vivo and propose suppressing PCR as a simple modification of a popular assay to monitor spacer acquisition with increased sensitivity.

RevDate: 2020-02-02

Mohammadinejad R, Biagioni A, Arunkumar G, et al (2020)

EMT signaling: potential contribution of CRISPR/Cas gene editing.

Cellular and molecular life sciences : CMLS pii:10.1007/s00018-020-03449-3 [Epub ahead of print].

Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process.

RevDate: 2020-02-14

Kaur N, Alok A, Shivani , et al (2020)

CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit.

Metabolic engineering, 59:76-86 pii:S1096-7176(20)30033-1 [Epub ahead of print].

Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the β-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCYε) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCYε gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCYε (GN-LCYε). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of β-carotene content up to 6-fold (~24 μg/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and α-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.

RevDate: 2020-02-11

Zhu J, Zhou X, Huang X, et al (2020)

Bacterial expression, purification, and initial characterization of a full-length Cas13b protein from Porphyromonas gingivalis.

Protein expression and purification, 169:105588 pii:S1046-5928(19)30467-X [Epub ahead of print].

The CRISPR-Cas13b system is a recently identified Class 2, RNA-targeting CRISPR-Cas system. The system has been repurposed to achieve robust mRNA knockdown and precise RNA-editing in mammalian cells. While the CRISPR-Cas13b system has become a powerful tool for nucleic acids manipulation, the mechanisms of the system are still not fully understood. Cas13b endonucleases from different bacterial species show poor overall sequence homologies, suggesting that structural (and probably functional) diversities may exist. It is therefore important to study CRISPR-Cas13b cases from different bacterial species. Here we report the expression, purification, and initial characterization of a Cas13b endonuclease that is associated with the 8th putative CRISPR locus from Porphyromonas gingivalis genome (Pgi8Cas13b). The full-length Pgi8Cas13b protein (1119 residues) was successfully expressed in E. Coli cells, and purified by affinity and ion-exchange chromatography methods. The purified protein is biologically active, being able to bind its cognate crRNA with high specificity and affinity. Preparation of biologically active Pgi8Cas13b protein provides the basis for further in vitro biochemical and biophysical studies of the Pgi8Cas13b CRISPR system.

RevDate: 2020-02-01

Zhang X, Cheng C, Sun W, et al (2020)

Engineering T Cells Using CRISPR/Cas9 for Cancer Therapy.

Methods in molecular biology (Clifton, N.J.), 2115:419-433.

Recent advances in the development of gene editing technologies, especially the CRISPR/Cas 9 system, have substantially enhanced our ability to make precise and efficient changes in the genomes of various cells. In particular, the genetic engineering of T cells holds huge potential to improve the efficacy and safety of T cells-based cancer therapy. Due to its ease of use and high efficiency, CRISPR/Cas9 enables efficient gene knockout, site-specific knock-in, and genome-wide screen in T cells. Here we review the current progress of applying gene editing to T-cell therapy, focusing on the technical aspects of the CRISPR/Cas9 platform. We also discuss the challenges and future prospects.

RevDate: 2020-02-01

Lennox KA, MA Behlke (2020)

Chemical Modifications in RNA Interference and CRISPR/Cas Genome Editing Reagents.

Methods in molecular biology (Clifton, N.J.), 2115:23-55.

Chemically modified oligonucleotides (ONs) are routinely used in the laboratory to assess gene function, and clinical advances are rapidly progressing as continual efforts are being made to optimize ON efficacy. Over the years, RNA interference (RNAi) has become one of the main tools used to inhibit RNA expression across a wide variety of species. Efforts have been made to improve the exogenous delivery of the double-stranded RNA components to the endogenous intracellular RNAi machinery to direct efficacious degradation of a user-defined RNA target. More recently, synthetic RNA ONs are being used to mimic the bacterial-derived CRISPR/Cas system to direct specific editing of the mammalian genome. Both of these techniques rely on the use of various chemical modifications to the RNA phosphate backbone or sugar in specific positions throughout the ONs to improve the desired biological outcome. Relevant chemical modifications also include conjugated targeting ligands to assist ON delivery to specific cell types. Chemical modifications are most beneficial for therapeutically relevant ONs, as they serve to enhance target binding, increase drug longevity, facilitate cell-specific targeting, improve internalization into productive intracellular compartments, and mitigate both sequence-specific as well as immune-related off-target effects (OTEs). The knowledge gained from years of optimizing RNAi reagents and characterizing the biochemical and biophysical properties of each chemical modification will hopefully accelerate the CRISPR/Cas technology into the clinic, as well as further expand the use of RNAi to treat currently undruggable diseases. This review discusses the most commonly employed chemical modifications in RNAi reagents and CRISPR/Cas guide RNAs and provides an overview of select publications that have demonstrated success in improving ON efficacy and/or mitigating undesired OTEs.

RevDate: 2020-02-04

Tan J, Zhang F, Karcher D, et al (2020)

Expanding the genome-targeting scope and the site selectivity of high-precision base editors.

Nature communications, 11(1):629.

Base editors (BEs) are RNA-guided CRISPR-Cas-derived genome editing tools that induce single-nucleotide changes. The limitations of current BEs lie in their low precision (especially when multiple target nucleotides of the deaminase are present within the activity window) and their restriction to targets that are in proper distance from the PAM sequence. We have recently developed high-precision cytidine BEs by engineering CDA1 truncations and nCas9 fusions that predominantly edit nucleotide C-18 relative to the PAM sequence NGG. Here, by testing fusions with Cas9 variants that recognize alternative PAMs, we provide a series of high-precision BEs that greatly expand the versatility of base editing. In addition, we obtained BEs that selectively edit C-15 or C-16. We also show that our high-precision BEs can substantially reduce off-target effect. These improved base editing tools will be widely applicable in basic research, biotechnology and gene therapy.

RevDate: 2020-02-06

Loriato VAP, Martins LGC, Euclydes NC, et al (2020)

Engineering resistance against geminiviruses: A review of suppressed natural defenses and the use of RNAi and the CRISPR/Cas system.

Plant science : an international journal of experimental plant biology, 292:110410.

The Geminiviridae family is one of the most successful and largest families of plant viruses that infect a large variety of important dicotyledonous and monocotyledonous crops and cause significant yield losses worldwide. This broad spectrum of host range is only possible because geminiviruses have evolved sophisticated strategies to overcome the arsenal of antiviral defenses in such diverse plant species. In addition, geminiviruses evolve rapidly through recombination and pseudo-recombination to naturally create a great diversity of virus species with divergent genome sequences giving the virus an advantage over the host recognition system. Therefore, it is not surprising that efficient molecular strategies to combat geminivirus infection under open field conditions have not been fully addressed. In this review, we present the anti-geminiviral arsenal of plant defenses, the evolved virulence strategies of geminiviruses to overcome these plant defenses and the most recent strategies that have been engineered for transgenic resistance. Although, the in vitro reactivation of suppressed natural defenses as well as the use of RNAi and CRISPR/Cas systems hold the potential for achieving broad-range resistance and/or immunity, potential drawbacks have been associated with each case.

RevDate: 2020-02-03

Farr L, Ghosh S, Jiang N, et al (2020)

CD74 signaling links inflammation to intestinal epithelial cell regeneration and promotes mucosal healing.

Cellular and molecular gastroenterology and hepatology pii:S2352-345X(20)30016-3 [Epub ahead of print].

BACKGROUND AND AIMS: The inflammatory response to intestinal damage promotes healing through mechanisms that are incompletely understood. Gene expression of CD74, the receptor for cytokine MIF, is increased in patients with inflammatory bowel disease (IBD), yet, the role of CD74 signaling in intestinal inflammation remains poorly understood. The aim of this study was to determine the functional role of CD74 signaling in intestinal inflammation.

METHODS: We studied the characteristics of CD74 protein expression in human IBD, and experimental colitis. The functional role of CD74 signaling in the intestine was investigated using cellular models, wild type, CD74-/-, and BM chimera mice, neutralizing anti-CD74 antibodies, flow cytometry, immunohistochemistry, immunofluorescence, immunoblotting and CRISPR-Cas technology.

RESULTS: In IBD patients and experimental colitis, CD74 receptor protein expression was elevated in inflamed intestinal tissue, prominently in the crypt epithelial cells. Using distinct but complimentary chemical and non-chemically induced mouse models of colitis with genetic and antibody neutralization approaches, we found that CD74 signaling was necessary for gut repair. Mechanistically, we found that the MIF cytokine, which is also elevated in colitis, stimulated CD74 receptor, enhancing intestinal epithelial cell proliferation through activation of the Akt and ERK pathways. Our data also suggest that CD74 signaling in immune cells was not essential for mucosal healing.

CONCLUSIONS: CD74 signaling is strongly activated during intestinal inflammation and protects the host by promoting epithelial cell regeneration, healing and maintaining mucosal barrier integrity. Enhancing the CD74 pathway may represent a unique therapeutic strategy for promoting healing in IBD.

RevDate: 2020-01-31

Tang Y, Fu P, Zhou Y, et al (2020)

Absence of the type I-E CRISPR-Cas system in Klebsiella pneumoniae clonal complex 258 is associated with dissemination of IncF epidemic resistance plasmids in this clonal complex.

The Journal of antimicrobial chemotherapy pii:5719345 [Epub ahead of print].

BACKGROUND: The pandemics caused by MDR Klebsiella pneumoniae are mostly due to the global dissemination of high-risk clonal complex 258 (CC258) and related IncF epidemic plasmids. However, the factors leading to the epidemiological advantages of CC258-IncF linkage remain obscure. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas) systems, providing adaptive immunity against invading DNA, play an important role in the interactions between plasmids and hosts.

OBJECTIVES: To investigate the relationship between CRISPR-Cas systems and the high-risk linkage CC258-IncF.

METHODS: CRISPR-Cas loci were detected among 381 collected K. pneumoniae clinical isolates and 207 K. pneumoniae complete genomes available in GenBank. MLST was used to determine the genetic relatedness of these isolates. Nucleotide BLAST was used to search for protospacers on K. pneumoniae plasmids.

RESULTS: We observed an epidemic correlation between CRISPR-Cas loci, CC258 and IncF plasmids. Interestingly, most type I-E CRISPR-Cas systems identified carried spacers matching the backbone regions of IncF plasmids.

CONCLUSIONS: Our results suggest that the absence of type I-E CRISPR-Cas systems in K. pneumoniae CC258 is strongly associated with the dissemination of IncF epidemic plasmids, contributing to the global success of the international high-risk linkage CC258-IncF. Our findings provide new information regarding the dissemination and evolution of the high-risk linkage of K. pneumoniae CC258-IncF and pave the way for new strategies to address the problem of antibiotic resistance.

RevDate: 2020-02-02

Kamruzzaman M, JR Iredell (2019)

CRISPR-Cas System in Antibiotic Resistance Plasmids in Klebsiella pneumoniae.

Frontiers in microbiology, 10:2934.

CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is a microbial adaptive immune system involved in defense against different types of mobile genetic elements. CRISPR-Cas systems are usually found in bacterial and archaeal chromosomes but have also been reported in bacteriophage genomes and in a few mega-plasmids. Klebsiella pneumoniae is an important member of the Enterobacteriaceae with which they share a huge pool of antibiotic resistance genes, mostly via plasmids. CRISPR-Cas systems have been identified in K. pneumoniae chromosomes, but relatively little is known of CRISPR-Cas in the plasmids resident in this species. In this study, we searched for CRISPR-Cas system in 699 complete plasmid sequences (>50-kb) and 217 complete chromosomal sequences of K. pneumoniae from GenBank and analyzed the CRISPR-Cas systems and CRISPR spacers found in plasmids and chromosomes. We found a putative CRISPR-Cas system in the 44 plasmids from Klebsiella species and GenBank search also identified the identical system in three plasmids from other Enterobacteriaceae, with CRISPR spacers targeting different plasmid and chromosome sequences. 45 of 47 plasmids with putative type IV CRISPR had IncFIB replicon and 36 of them had an additional IncHI1B replicon. All plasmids except two are very large (>200 kb) and half of them carried multiple antibiotic resistance genes including blaCTX-M , blaNDM , blaOXA . To our knowledge, this is the first report of multi drug resistance plasmids from Enterobacteriaceae with their own CRISPR-Cas system and it is possible that the plasmid type IV CRISPR may depend on the chromosomal type I-E CRISPRs for their competence. Both chromosomal and plasmid CRISPRs target a large variety of plasmids from this species, further suggesting key roles in the epidemiology of large plasmids.

RevDate: 2020-02-05
CmpDate: 2020-02-05

Urnov FD (2020)

Prime Time for Genome Editing?.

The New England journal of medicine, 382(5):481-484.

RevDate: 2020-02-14

Gong T, Zeng J, Tang B, et al (2020)

CRISPR-Cas systems in oral microbiome: From immune defense to physiological regulation.

Molecular oral microbiology [Epub ahead of print].

The clustered regularly interspaced short palindromic repeats with CRISPR-associated proteins (CRISPR-Cas) system, found in bacteria and archaea, provides sequence-based adaptive immunity against mobile genetic elements, including phages and plasmids. The oral cavity contains approximately 700 prokaryote species harboring known CRISPR-Cas systems, including type I, type II, type III, type V, and type VI, and unidentified CRISPR-Cas systems. There is increasing evidence to suggest that different CRISPR-Cas systems in the human oral microbiome can affect bacterial physiology through different mechanisms. Here, we review the canonical and novel functions of the CRISPR-Cas system, including defense against the invasion of foreign mobile elements, biofilm formation, acquisition of resistance genes, DNA repair, regulation of interspecific competition and intraspecific diversification, stress responses, and gene expression regulation. Overall, the mechanisms involved in CRISPR-Cas systems and their effects on bacterial physiology provide new insights into our understanding of the function and application of methods (including gene editing, modulation of CRISPR-Cas by anti-CRISPR, antimicrobials) on the oral microbiome.

RevDate: 2020-02-15

Yan MY, Li SS, Ding XY, et al (2020)

A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis.

mBio, 11(1):.

New tools for genetic manipulation of Mycobacterium tuberculosis are needed for the development of new drug regimens and vaccines aimed at curing tuberculosis infections. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems generate a highly specific double-strand break at the target site that can be repaired via nonhomologous end joining (NHEJ), resulting in the desired genome alteration. In this study, we first improved the NHEJ repair pathway and developed a CRISPR-Cas-mediated genome-editing method that allowed us to generate markerless deletion in Mycobacterium smegmatis, Mycobacterium marinum, and M. tuberculosis Then, we demonstrated that this system could efficiently achieve simultaneous generation of double mutations and large-scale genetic mutations in M. tuberculosis Finally, we showed that the strategy we developed can also be used to facilitate genome editing in Escherichia coliIMPORTANCE The global health impact of M. tuberculosis necessitates the development of new genetic tools for its manipulation, to facilitate the identification and characterization of novel drug targets and vaccine candidates. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) genome editing has proven to be a powerful genetic tool in various organisms; to date, however, attempts to use this approach in M. tuberculosis have failed. Here, we describe a genome-editing tool based on CRISPR cleavage and the nonhomologous end-joining (NHEJ) repair pathway that can efficiently generate deletion mutants in M. tuberculosis More importantly, this system can generate simultaneous double mutations and large-scale genetic mutations in this species. We anticipate that this CRISPR-NHEJ-assisted genome-editing system will be broadly useful for research on mycobacteria, vaccine development, and drug target profiling.

RevDate: 2020-01-30

Sanor LD, Flowers GP, CM Crews (2020)

Multiplex CRISPR/Cas screen in regenerating haploid limbs of chimeric Axolotls.

eLife, 9:.

Axolotls and other salamanders can regenerate entire limbs after amputation as adults, and much recent effort has sought to identify the molecular programs controlling this process. While targeted mutagenesis approaches like CRISPR/Cas9 now permit gene-level investigation of these mechanisms, genetic screening in the axolotl requires an extensive commitment of time and space. Previously, we quantified CRISPR/Cas9-generated mutations in the limbs of mosaic mutant axolotls before and after regeneration and found that the regenerated limb is a highfidelity replicate of the original limb (Flowers et al. 2017). Here, we circumvent aforementioned genetic screening limitations and present methods for a multiplex CRISPR/Cas9 haploid screen in chimeric axolotls (MuCHaChA), which is a novel platform for haploid genetic screening in animals to identify genes essential for limb regeneration.

RevDate: 2020-01-28

Creutzburg SCA, Wu WY, Mohanraju P, et al (2020)

Good guide, bad guide: spacer sequence-dependent cleavage efficiency of Cas12a.

Nucleic acids research pii:5716457 [Epub ahead of print].

Genome editing has recently made a revolutionary development with the introduction of the CRISPR-Cas technology. The programmable CRISPR-associated Cas9 and Cas12a nucleases generate specific dsDNA breaks in the genome, after which host DNA-repair mechanisms can be manipulated to implement the desired editing. Despite this spectacular progress, the efficiency of Cas9/Cas12a-based engineering can still be improved. Here, we address the variation in guide-dependent efficiency of Cas12a, and set out to reveal the molecular basis of this phenomenon. We established a sensitive and robust in vivo targeting assay based on loss of a target plasmid encoding the red fluorescent protein (mRFP). Our results suggest that folding of both the precursor guide (pre-crRNA) and the mature guide (crRNA) have a major influence on Cas12a activity. Especially, base pairing of the direct repeat, other than with itself, was found to be detrimental to the activity of Cas12a. Furthermore, we describe different approaches to minimize base-pairing interactions between the direct repeat and the variable part of the guide. We show that design of the 3' end of the guide, which is not involved in target strand base pairing, may result in substantial improvement of the guide's targeting potential and hence of its genome editing efficiency.

RevDate: 2020-02-09

Hausmann C, Vogt A, Kerscher M, et al (2020)

Optimizing skin pharmacotherapy for older patients: the future is at hand but are we ready for it?.

Drug discovery today pii:S1359-6446(20)30037-4 [Epub ahead of print].

Age-related changes affect both the local pharmacotherapy of skin diseases and the transdermal administration of drugs. The development of aged skin models disregards the highly individual process of aging, facilitating general conclusions for older patients. Nevertheless, 'omics technology, high-content screening, and non-invasive imaging, as well as bioprinting, CRISPR-Cas, and, patients-on-a-chip, can retrieve personalized information for the generation of in vitro models. Herein, we suggest a strategy to optimize pharmacotherapy for older patients. The technology for relevant human cell-based models is at hand and the consideration of patient heterogeneity is required to unlock their full potential.

RevDate: 2020-01-28

Kaneko T, Y Nakagawa (2020)

Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos.

Cryobiology pii:S0011-2240(19)30615-7 [Epub ahead of print].

Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the "Technique for Animal Knockout system by Electroporation (TAKE)," has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.

RevDate: 2020-01-25

Nimkar S, B Anand (2020)

Cas3/I-C mediated target DNA recognition and cleavage during CRISPR interference are independent of the composition and architecture of Cascade surveillance complex.

Nucleic acids research pii:5715815 [Epub ahead of print].

In type I CRISPR-Cas system, Cas3-a nuclease cum helicase-in cooperation with Cascade surveillance complex cleaves the target DNA. Unlike the Cascade/I-E, which is composed of five subunits, the Cascade/I-C is made of only three subunits lacking the CRISPR RNA processing enzyme Cas6, whose role is assumed by Cas5. How these differences in the composition and organization of Cascade subunits in type I-C influence the Cas3/I-C binding and its target cleavage mechanism is poorly understood. Here, we show that Cas3/I-C is intrinsically a single-strand specific promiscuous nuclease. Apart from the helicase domain, a constellation of highly conserved residues-which are unique to type I-C-located in the uncharacterized C-terminal domain appears to influence the nuclease activity. Recruited by Cascade/I-C, the HD nuclease of Cas3/I-C nicks the single-stranded region of the non-target strand and positions the helicase motor. Powered by ATP, the helicase motor reels in the target DNA, until it encounters the roadblock en route, which stimulates the HD nuclease. Remarkably, we show that Cas3/I-C supplants Cas3/I-E for CRISPR interference in type I-E in vivo, suggesting that the target cleavage mechanism is evolutionarily conserved between type I-C and type I-E despite the architectural difference exhibited by Cascade/I-C and Cascade/I-E.

RevDate: 2020-01-30

Sanderson H, Ortega-Polo R, Zaheer R, et al (2020)

Comparative genomics of multidrug-resistant Enterococcus spp. isolated from wastewater treatment plants.

BMC microbiology, 20(1):20.

BACKGROUND: Wastewater treatment plants (WWTPs) are considered hotspots for the environmental dissemination of antimicrobial resistance (AMR) determinants. Vancomycin-Resistant Enterococcus (VRE) are candidates for gauging the degree of AMR bacteria in wastewater. Enterococcus faecalis and Enterococcus faecium are recognized indicators of fecal contamination in water. Comparative genomics of enterococci isolated from conventional activated sludge (CAS) and biological aerated filter (BAF) WWTPs was conducted.

RESULTS: VRE isolates, including E. faecalis (n = 24), E. faecium (n = 11), E. casseliflavus (n = 2) and E. gallinarum (n = 2) were selected for sequencing based on WWTP source, species and AMR phenotype. The pangenomes of E. faecium and E. faecalis were both open. The genomic fraction related to the mobilome was positively correlated with genome size in E. faecium (p < 0.001) and E. faecalis (p < 0.001) and with the number of AMR genes in E. faecium (p = 0.005). Genes conferring vancomycin resistance, including vanA and vanM (E. faecium), vanG (E. faecalis), and vanC (E. casseliflavus/E. gallinarum), were detected in 20 genomes. The most prominent functional AMR genes were efflux pumps and transporters. A minimum of 16, 6, 5 and 3 virulence genes were detected in E. faecium, E. faecalis, E. casseliflavus and E. gallinarum, respectively. Virulence genes were more common in E. faecalis and E. faecium, than E. casseliflavus and E. gallinarum. A number of mobile genetic elements were shared among species. Functional CRISPR/Cas arrays were detected in 13 E. faecalis genomes, with all but one also containing a prophage. The lack of a functional CRISPR/Cas arrays was associated with multi-drug resistance in E. faecium. Phylogenetic analysis demonstrated differential clustering of isolates based on original source but not WWTP. Genes related to phage and CRISPR/Cas arrays could potentially serve as environmental biomarkers.

CONCLUSIONS: There was no discernible difference between enterococcal genomes from the CAS and BAF WWTPs. E. faecalis and E. faecium have smaller genomes and harbor more virulence, AMR, and mobile genetic elements than other Enterococcus spp.

RevDate: 2020-02-07

Bauer R, Neffgen N, Grempels A, et al (2020)

Heterogeneity of Streptococcus anginosus ß-hemolysis in relation to CRISPR/Cas.

Molecular oral microbiology [Epub ahead of print].

Streptococcus anginosus is a commensal of the oral mucosa that can cause severe invasive infections. A considerable proportion of Streptococcus anginosus strains are ß-hemolytic due to the presence of an SLS-like gene cluster. However, the majority of strains do not display ß-hemolysis. To investigate ß-hemolysin heterogeneity in S. anginosus, we determined the presence of sag genes and correlated it with the presence of CRISPR/Cas genes in a collection of ß-hemolytic and non-ß-hemolytic strains. All of the ß-hemolytic strains carried the sag gene cluster. In contrast to other streptococci, clinical S. anginosus strains that do not display ß-hemolysis do not harbor sag genes. Phylogenetic analysis of the ß-hemolytic strains revealed that they belong to two previously defined clusters within S. anginosus. Correlation with CRISPR/Cas genes showed a significant difference for the presence of CRISPR/Cas in ß-hemolytic versus non-ß-hemolytic isolates. The presence of the CRISPR/Cas type IIA or type IIC locus is associated with the absence of sag genes; in 65% of the non-ß-hemolytic strains a CRISPR/Cas locus was found, while only 24% of ß-hemolytic strains carry CRISPR/Cas genes. Further analysis of the spacer content of the CRISPR systems revealed the presence of multiple self-targeting sequences directed against S. anginosus genes. These results support the hypothesis that horizontal gene transfer is involved in the acquisition of ß-hemolysin genes and that CRISPR/Cas may limit DNA uptake in S. anginosus.

RevDate: 2020-01-24

Golchin A, Shams F, F Karami (2020)

Advancing Mesenchymal Stem Cell Therapy with CRISPR/Cas9 for Clinical Trial Studies.

Advances in experimental medicine and biology [Epub ahead of print].

Currently, regenerative medicine and cellular-based therapy have been in the center of attention worldwide in advanced medical technology. Mesenchymal stem cell (MSC) as a suitable stem cell source for cell-based therapy has been shown to be safe and effective in multiple clinical trial studies (CTSs) of several diseases. Despite the advantages, MSC needs more investigation to enhance its therapeutic application. The CRISPR/Cas system is a novel technique for editing of genes that is being explored as a means to improve MSCs therapeutic usage. In this study, we review the recent studies that explore CRISPR potency in gene engineering of MSCs, which have great relevance in MSC-based therapies. However, CRISPR/Cas technology make possible specific targeting of loci in target genes, but next-generation MSC-based therapies to achieve extensive clinical application need dedicated efforts.

RevDate: 2020-02-13

Rollie C, Chevallereau A, Watson BNJ, et al (2020)

Targeting of temperate phages drives loss of type I CRISPR-Cas systems.

Nature, 578(7793):149-153.

On infection of their host, temperate viruses that infect bacteria (bacteriophages; hereafter referred to as phages) enter either a lytic or a lysogenic cycle. The former results in lysis of bacterial cells and phage release (resulting in horizontal transmission), whereas lysogeny is characterized by the integration of the phage into the host genome, and dormancy (resulting in vertical transmission)1. Previous co-culture experiments using bacteria and mutants of temperate phages that are locked in the lytic cycle have shown that CRISPR-Cas systems can efficiently eliminate the invading phages2,3. Here we show that, when challenged with wild-type temperate phages (which can become lysogenic), type I CRISPR-Cas immune systems cannot eliminate the phages from the bacterial population. Furthermore, our data suggest that, in this context, CRISPR-Cas immune systems are maladaptive to the host, owing to the severe immunopathological effects that are brought about by imperfect matching of spacers to the integrated phage sequences (prophages). These fitness costs drive the loss of CRISPR-Cas from bacterial populations, unless the phage carries anti-CRISPR (acr) genes that suppress the immune system of the host. Using bioinformatics, we show that this imperfect targeting is likely to occur frequently in nature. These findings help to explain the patchy distribution of CRISPR-Cas immune systems within and between bacterial species, and highlight the strong selective benefits of phage-encoded acr genes for both the phage and the host under these circumstances.

RevDate: 2020-02-05

Glover L (2020)

mSphere of Influence: Expanding the CRISPR Sphere with Single-Locus Proteomics.

mSphere, 5(1):.

Lucy Glover's research focuses on the role of DNA repair and recombination in antigenic variation in the parasite Trypanosoma brucei, the causative agent of both human and animal African trypanosomiasis. In this mSphere of Influence article, she reflects on how "A CRISPR-based approach for proteomic analysis of a single genomic locus" by Z. J. Waldrip, S. D. Byrum, A. J. Storey, J. Gao, et al. (Epigenetics 9:1207-1211, 2014, https://doi.org/10.4161/epi.29919) made an impact on her research by taking the precision of CRISPR-Cas9 and repurposing it to look at single-locus proteomics. By using this technology in trypanosomes, Dr. Glover and her colleagues could study the dynamic accumulation of repair proteins after specific damage and gain insight into how the location of a double-strand break (DSB) dictates repair pathway choice and how this may influence immune evasion in these parasites.

RevDate: 2020-01-22

Adamson CS, MM Nevels (2020)

Bright and Early: Inhibiting Human Cytomegalovirus by Targeting Major Immediate-Early Gene Expression or Protein Function.

Viruses, 12(1): pii:v12010110.

The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved antivirals come with major limitations. Most approved HCMV antivirals target late molecular processes in the viral replication cycle including DNA replication and packaging. "Bright and early" events in HCMV infection have not been exploited for systemic prevention or treatment of disease. Initiation of HCMV replication depends on transcription from the viral major immediate-early (IE) gene. Alternative transcripts produced from this gene give rise to the IE1 and IE2 families of viral proteins, which localize to the host cell nucleus. The IE1 and IE2 proteins are believed to control all subsequent early and late events in HCMV replication, including reactivation from latency, in part by antagonizing intrinsic and innate immune responses. Here we provide an update on the regulation of major IE gene expression and the functions of IE1 and IE2 proteins. We will relate this insight to experimental approaches that target IE gene expression or protein function via molecular gene silencing and editing or small chemical inhibitors.

RevDate: 2020-02-10
CmpDate: 2020-02-10

Liu PF, Q Wu (2020)

Probing 3D genome by CRISPR/Cas9.

Yi chuan = Hereditas, 42(1):18-31.

CRISPR/Cas9 system has significant advantages in gene editing strategy, offering cost-effective and efficient means to modify and edit the genomes of animals, plants, and microorganisms. Three-dimensional (3D) genome is an emerging and interdisciplinary field catapulted by combined technological breakthroughs of chromosome conformation capture with next-generation sequencing and live imaging with super-resolution microscopy. An important aspect of 3D genomics is to model structural variations and label specific genomic fragments to investigate the effects of manipulation of genomic elements on gene expression regulation, cell development and differentiation, and spatial location of chromosomal regions. Therefore, CRISPR/Cas9 system and its derivative technologies of DNA-fragment editing are excellent toolboxes for investigating dynamics and functions of the higher-order chromatin organization and three-dimensional genome structure. In this review, we describe the opportunities and challenges of CRISPR as well as its derivative technologies in 3D genome research, thereby providing some critical references and future research directions in the field.

RevDate: 2020-01-28

Beisel CL (2020)

Methods for characterizing, applying, and teaching CRISPR-Cas systems.

RevDate: 2020-02-03
CmpDate: 2020-02-03

Mohammadinejad R, Sassan H, Pardakhty A, et al (2020)

ZEB1 and ZEB2 gene editing mediated by CRISPR/Cas9 in A549 cell line.

Bratislavske lekarske listy, 121(1):31-36.

OBJECTIVES: One of the best approaches for recognition of protein function is the induction of mutations for a gene knockout. In line with this strategy, gene editing tools allow researchers to induce these mutations. Lung cancer is one of the leading causes of death worldwide. ZEB1 and ZEB2 genes are the candidates for this disease.

METHODS: The ZEB1 and ZEB2 knockout in the non-small cell lung cancer cell line (A549 cell) was investigated. Purification of recombination plasmids was performed from bacteria and then was transported to the A549 cell line. The deletion of ZEB1 and ZEB2 were examined by PCR.

RESULTS: The results demonstrated the mutation and deletion in ZEB1 and ZEB2 genes. Based on the findings of this study, A549 cells were transfected with the vectors carrying the sgRNA/Cas9, simultaneously. The DNA fragment demonstrated the presence of indels in target sites as well as provided the potential of CRISPR/Cas9 system.

CONCLUSION: CRISPR/Cas9 offers a great potential as an efficient technique for editing of ZEB1 and ZEB2 genes in A549 cell line (Tab. 1, Fig. 6, Ref. 44).

RevDate: 2020-02-06

Jia Y, Yang B, Ross P, et al (2020)

Comparative Genomics Analysis of Lactobacillus mucosae from Different Niches.

Genes, 11(1): pii:genes11010095.

The potential probiotic benefits of Lactobacillus mucosae have received increasing attention. To investigate the genetic diversity of L. mucosae, comparative genomic analyses of 93 strains isolated from different niches (human and animal gut, human vagina, etc.) and eight strains of published genomes were conducted. The results showed that the core genome of L. mucosae mainly encoded translation and transcription, amino acid biosynthesis, sugar metabolism, and defense function while the pan-genomic curve tended to be close. The genetic diversity of L. mucosae mainly reflected in carbohydrate metabolism and immune/competitive-related factors, such as exopolysaccharide (EPS), enterolysin A, and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas. It was worth noting that this research firstly predicted the complete EPS operon shared among L. mucosae. Additionally, the type IIIA CRISPR-Cas system was discovered in L. mucosae for the first time. This work provided new ideas for the study of this species.

RevDate: 2020-01-16

Palumbo CM, Gutierrez-Bujari JM, O'Geen H, et al (2020)

Versatile 3' Functionalization of CRISPR Single Guide RNA.

Chembiochem : a European journal of chemical biology [Epub ahead of print].

Specific applications of CRISPR/Cas genome editing systems benefit from chemical modifications of the sgRNA. Here we describe a versatile and efficient strategy for functionalization of the 3' end of a sgRNA. An exemplary collection of six chemically modified sgRNAs was prepared containing crosslinkers, a fluorophore and biotin. Modification of the sgRNA 3' end was broadly tolerated by S. pyogenes Cas9 in an in vitro DNA cleavage assay. The 3'-biotinylated sgRNA was used as an affinity reagent to identify IGF2BP1, YB1 and hnRNP K as sgRNA-binding proteins present in HEK293T cells. Overall, the modification strategy presented here has the potential to expand on current applications of CRISPR/Cas systems.

RevDate: 2020-01-16

Hao Y, Zong W, Zeng D, et al (2020)

Shortened snRNA promoters for efficient CRISPR/Cas-based multiplex genome editing in monocot plants.

RevDate: 2020-01-21

Hampton HG, Watson BNJ, PC Fineran (2020)

The arms race between bacteria and their phage foes.

Nature, 577(7790):327-336.

Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.

RevDate: 2020-01-16

Yang D, Wang Z, Ma J, et al (2020)

Glycine Cleavage System and cAMP Receptor Protein Co-Regulate CRISPR/cas3 Expression to Resist Bacteriophage.

Viruses, 12(1): pii:v12010090.

The CRISPR/Cas system protects bacteria against bacteriophage and plasmids through a sophisticated mechanism where cas operon plays a crucial role consisting of cse1 and cas3. However, comprehensive studies on the regulation of cas3 operon of the Type I-E CRISPR/Cas system are scarce. Herein, we investigated the regulation of cas3 in Escherichia coli. The mutation in gcvP or crp reduced the CRISPR/Cas system interference ability and increased bacterial susceptibility to phage, when the casA operon of the CRISPR/Cas system was activated. The silence of the glycine cleavage system (GCS) encoded by gcvTHP operon reduced cas3 expression. Adding N5, N10-methylene tetrahydrofolate (N5, N10-mTHF), which is the product of GCS-catalyzed glycine, was able to activate cas3 expression. In addition, a cAMP receptor protein (CRP) encoded by crp activated cas3 expression via binding to the cas3 promoter in response to cAMP concentration. Since N5, N10-mTHF provides one-carbon unit for purine, we assumed GCS regulates cas3 through associating with CRP. It was evident that the mutation of gcvP failed to further reduce the cas3 expression with the crp deletion. These results illustrated a novel regulatory pathway which GCS and CRP co-regulate cas3 of the CRISPR/Cas system and contribute to the defence against invasive genetic elements, where CRP is indispensable for GCS regulation of cas3 expression.

RevDate: 2020-02-04

Weissman JL, PLF Johnson (2020)

Network-Based Prediction of Novel CRISPR-Associated Genes in Metagenomes.

mSystems, 5(1):.

A diversity of clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity to bacteria and archaea through recording "memories" of past viral infections. Recently, many novel CRISPR-associated proteins have been discovered via computational studies, but those studies relied on biased and incomplete databases of assembled genomes. We avoided these biases and applied a network theory approach to search for novel CRISPR-associated genes by leveraging subtle ecological cooccurrence patterns identified from environmental metagenomes. We validated our method using existing annotations and discovered 32 novel CRISPR-associated gene families. These genes span a range of putative functions, with many potentially regulating the response to infection.IMPORTANCE Every branch on the tree of life, including microbial life, faces the threat of viral pathogens. Over the course of billions of years of coevolution, prokaryotes have evolved a great diversity of strategies to defend against viral infections. One of these is the CRISPR adaptive immune system, which allows microbes to "remember" past infections in order to better fight them in the future. There has been much interest among molecular biologists in CRISPR immunity because this system can be repurposed as a tool for precise genome editing. Recently, a number of comparative genomics approaches have been used to detect novel CRISPR-associated genes in databases of genomes with great success, potentially leading to the development of new genome-editing tools. Here, we developed novel methods to search for these distinct classes of genes directly in environmental samples ("metagenomes"), thus capturing a more complete picture of the natural diversity of CRISPR-associated genes.

RevDate: 2020-01-15

Deecker SR, AW Ensminger (2020)

Type I-F CRISPR-Cas Distribution and Array Dynamics in Legionella pneumophila.

G3 (Bethesda, Md.) pii:g3.119.400813 [Epub ahead of print].

In bacteria and archaea, several distinct types of CRISPR-Cas systems provide adaptive immunity through broadly similar mechanisms: short nucleic acid sequences derived from foreign DNA, known as spacers, engage in complementary base pairing with invasive genetic elements setting the stage for nucleases to degrade the target DNA. A hallmark of type I CRISPR-Cas systems is their ability to acquire spacers in response to both new and previously encountered invaders (naïve and primed acquisition, respectively). Our phylogenetic analyses of 43 L. pneumophila type I-F CRISPR-Cas systems and their resident genomes suggest that many of these systems have been horizontally acquired. These systems are frequently encoded on plasmids and can co-occur with nearly identical chromosomal loci. We show that two such co-occurring systems are highly protective and undergo efficient primed acquisition in the lab. Furthermore, we observe that targeting by one system's array can prime spacer acquisition in the other. Lastly, we provide experimental and genomic evidence for a model in which primed acquisition can efficiently replenish a depleted type I CRISPR array following a mass spacer deletion event.

RevDate: 2020-01-15

Cui L, Wang X, Huang D, et al (2020)

CRISPR-cas3 of Salmonella Upregulates Bacterial Biofilm Formation and Virulence to Host Cells by Targeting Quorum-Sensing Systems.

Pathogens (Basel, Switzerland), 9(1): pii:pathogens9010053.

Salmonella is recognized as one of the most common microbial pathogens worldwide. The bacterium contains the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, providing adaptive immunity against invading foreign nucleic acids. Previous studies suggested that certain bacteria employ the Cas proteins of CRISPR-Cas systems to target their own genes, which also alters the virulence during invasion of mammals. However, whether CRISPR-Cas systems in Salmonella have similar functions during bacterial invasion of host cells remains unknown. Here, we systematically analyzed the genes that are regulated by Cas3 in a type I-E CRISPR-Cas system and the virulence changes due to the deletion of cas3 in Salmonella enterica serovar Enteritidis. Compared to the cas3 gene wild-type (cas3 WT) Salmonella strain, cas3 deletion upregulated the lsrFGBE genes in lsr (luxS regulated) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and Salmonella pathogenicity island 1 (SPI-1) genes related to the type three secretion system (T3SS). Consistently, the biofilm formation ability was downregulated in the cas3 deletion mutant (Δcas3). The bacterial invasive and intracellular capacity of Δcas3 to host cells was also reduced, thereby increasing the survival of infected host cells and live chickens. By the transcriptome-wide screen (RNA-Seq), we found that the cas3 gene impacts a series of genes related to QS, the flagellum, and SPI-1-T3SS system, thereby altering the virulence phenotypes. As QS SPI-1-T3SS and CRISPR-Cas systems are widely distributed in the bacteria kingdom, our findings extend our understanding of virulence regulation and pathogenicity in mammalian hosts for Salmonella and potentially other bacteria.

RevDate: 2020-01-15

Wang S, Yang B, Ross RP, et al (2020)

Comparative Genomics Analysis of Lactobacillus ruminis from Different Niches.

Genes, 11(1): pii:genes11010070.

Lactobacillus ruminis is a commensal motile lactic acid bacterium living in the intestinal tract of humans and animals. Although a few genomes of L. ruminis were published, most of them were animal derived. To explore the genetic diversity and potential niche-specific adaptation changes of L. ruminis, in the current work, draft genomes of 81 L. ruminis strains isolated from human, bovine, piglet, and other animals were sequenced, and comparative genomic analysis was performed. The genome size and GC content of L. ruminis on average were 2.16 Mb and 43.65%, respectively. Both the origin and the sampling distance of these strains had a great influence on the phylogenetic relationship. For carbohydrate utilization, the human-derived L. ruminis strains had a higher consistency in the utilization of carbon source compared to the animal-derived strains. L. ruminis mainly increased the competitiveness of niches by producing class II bacteriocins. The type of clustered regularly interspaced short palindromic repeats /CRISPR-associated (CRISPR/Cas) system presented in L. ruminis was mainly subtype IIA. The diversity of CRISPR/Cas locus depended on the high denaturation of spacer number and sequence, although cas1 protein was relatively conservative. The genetic differences in those newly sequenced L. ruminis strains highlighted the gene gains and losses attributed to niche adaptations.

RevDate: 2020-01-17

Li H, Yang Y, Hong W, et al (2020)

Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects.

Signal transduction and targeted therapy, 5:1.

Based on engineered or bacterial nucleases, the development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all eukaryotic cells. Genome editing has extended our ability to elucidate the contribution of genetics to disease by promoting the creation of more accurate cellular and animal models of pathological processes and has begun to show extraordinary potential in a variety of fields, ranging from basic research to applied biotechnology and biomedical research. Recent progress in developing programmable nucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas-associated nucleases, has greatly expedited the progress of gene editing from concept to clinical practice. Here, we review recent advances of the three major genome editing technologies (ZFNs, TALENs, and CRISPR/Cas9) and discuss the applications of their derivative reagents as gene editing tools in various human diseases and potential future therapies, focusing on eukaryotic cells and animal models. Finally, we provide an overview of the clinical trials applying genome editing platforms for disease treatment and some of the challenges in the implementation of this technology.

RevDate: 2020-02-04

Lau RK, Ye Q, Birkholz EA, et al (2020)

Structure and Mechanism of a Cyclic Trinucleotide-Activated Bacterial Endonuclease Mediating Bacteriophage Immunity.

Molecular cell pii:S1097-2765(19)30923-2 [Epub ahead of print].

Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.

RevDate: 2020-02-12

Weng Y, Huang Q, Li C, et al (2019)

Improved Nucleic Acid Therapy with Advanced Nanoscale Biotechnology.

Molecular therapy. Nucleic acids, 19:581-601 pii:S2162-2531(19)30399-3 [Epub ahead of print].

Due to a series of systemic and intracellular obstacles in nucleic acid (NA) therapy, including fast degradation in blood, renal clearance, poor cellular uptake, and inefficient endosomal escape, NAs may need delivery methods to transport to the cell nucleus or cytosol to be effective. Advanced nanoscale biotechnology-associated strategies, such as controlling the particle size, charge, drug loading, response to environmental signals, or other physical/chemical properties of delivery carriers, have provided great help for the in vivo and in vitro delivery of NA therapeutics. In this review, we introduce the characteristics of different NA modalities and illustrate how advanced nanoscale biotechnology assists NA therapy. The specific features and challenges of various nanocarriers in clinical and preclinical studies are summarized and discussed. With the help of advanced nanoscale biotechnology, some of the major barriers to the development of NA therapy will eventually be overcome in the near future.

RevDate: 2020-01-13

Wang R, Angenent GC, Seymour G, et al (2020)

Revisiting the Role of Master Regulators in Tomato Ripening.

Trends in plant science pii:S1360-1385(19)30305-X [Epub ahead of print].

The study of transcriptional regulation of tomato ripening has been led by spontaneous mutations in transcription factor (TF) genes that completely inhibit normal ripening, suggesting that they are 'master regulators'. Studies using CRISPR/Cas9 mutagenesis to produce knockouts of the underlying genes indicate a different picture, suggesting that the regulation is more robust than previously thought. This requires us to revisit our model of the regulation of ripening and replace it with one involving a network of partially redundant components. At the same time, the fast rise of CRISPR/Cas mutagenesis, resulting in unexpectedly weak phenotypes, compared with knockdown technology, suggests that compensatory mechanisms may obscure protein functions. This emphasises the need for assessment of these mechanisms in plants and for the careful design of mutagenesis experiments.

RevDate: 2020-02-15

Jia N, Xie W, de la Cruz MJ, et al (2020)

Structure-function insights into the initial step of DNA integration by a CRISPR-Cas-Transposon complex.

Cell research, 30(2):182-184.

RevDate: 2020-01-10

Hidalgo-Cantabrana C, R Barrangou (2020)

Characterization and applications of Type I CRISPR-Cas systems.

Biochemical Society transactions pii:221842 [Epub ahead of print].

CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.

RevDate: 2020-01-13

Wesseler J, Politiek H, D Zilberman (2019)

The Economics of Regulating New Plant Breeding Technologies - Implications for the Bioeconomy Illustrated by a Survey Among Dutch Plant Breeders.

Frontiers in plant science, 10:1597.

New plant breeding technologies (NPBTs) are increasingly used for developing new plants with novel traits. The science tells us that those plants in general are as safe as than those once developed using "conventional" plant breeding methods. The knowledge about the induced changes and properties of the new plants by using NPBTs is more precise. This should lead to the conclusion that plants developed using NPBTs should not be regulated differently than those developed using "conventional" plant breeding methods. This contribution discusses the economics of regulating new plant breeding technologies. We first develop the theoretical model and elaborate on the different regulatory approaches being used and compare their advantages and disadvantages. Then we provide a perspectives on EU regulation around mutagenesis-based New Plant Breeding Techniques (NPBT), formed by new insights from a survey among Dutch plant breeding companies. The survey measures the attitude of breeding companies towards the ruling of the EU Court of Justice that subjected the use of CRISPR-Cas in the development of new plant varieties under the general EU regulations around GMOs. The results show that plant breeders experience a financial barrier because of the ruling, with perceived negative impact on competitiveness and investments in CRISPR-Cas as a result. The degree of negative impact differs however significantly among seed-sectors and company sizes. One of the most striking results was the relative optimism of companies in the sector about more lenient legislation in the next five years, despite the stated negative effects.

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RJR Experience and Expertise

Researcher

Robbins holds BS, MS, and PhD degrees in the life sciences. He served as a tenured faculty member in the Zoology and Biological Science departments at Michigan State University. He is currently exploring the intersection between genomics, microbial ecology, and biodiversity — an area that promises to transform our understanding of the biosphere.

Educator

Robbins has extensive experience in college-level education: At MSU he taught introductory biology, genetics, and population genetics. At JHU, he was an instructor for a special course on biological database design. At FHCRC, he team-taught a graduate-level course on the history of genetics. At Bellevue College he taught medical informatics.

Administrator

Robbins has been involved in science administration at both the federal and the institutional levels. At NSF he was a program officer for database activities in the life sciences, at DOE he was a program officer for information infrastructure in the human genome project. At the Fred Hutchinson Cancer Research Center, he served as a vice president for fifteen years.

Technologist

Robbins has been involved with information technology since writing his first Fortran program as a college student. At NSF he was the first program officer for database activities in the life sciences. At JHU he held an appointment in the CS department and served as director of the informatics core for the Genome Data Base. At the FHCRC he was VP for Information Technology.

Publisher

While still at Michigan State, Robbins started his first publishing venture, founding a small company that addressed the short-run publishing needs of instructors in very large undergraduate classes. For more than 20 years, Robbins has been operating The Electronic Scholarly Publishing Project, a web site dedicated to the digital publishing of critical works in science, especially classical genetics.

Speaker

Robbins is well-known for his speaking abilities and is often called upon to provide keynote or plenary addresses at international meetings. For example, in July, 2012, he gave a well-received keynote address at the Global Biodiversity Informatics Congress, sponsored by GBIF and held in Copenhagen. The slides from that talk can be seen HERE.

Facilitator

Robbins is a skilled meeting facilitator. He prefers a participatory approach, with part of the meeting involving dynamic breakout groups, created by the participants in real time: (1) individuals propose breakout groups; (2) everyone signs up for one (or more) groups; (3) the groups with the most interested parties then meet, with reports from each group presented and discussed in a subsequent plenary session.

Designer

Robbins has been engaged with photography and design since the 1960s, when he worked for a professional photography laboratory. He now prefers digital photography and tools for their precision and reproducibility. He designed his first web site more than 20 years ago and he personally designed and implemented this web site. He engages in graphic design as a hobby.

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CRISPR-Cas

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

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E-mail: RJR8222@gmail.com

Collection of publications by R J Robbins

Reprints and preprints of publications, slide presentations, instructional materials, and data compilations written or prepared by Robert Robbins. Most papers deal with computational biology, genome informatics, using information technology to support biomedical research, and related matters.

Research Gate page for R J Robbins

ResearchGate is a social networking site for scientists and researchers to share papers, ask and answer questions, and find collaborators. According to a study by Nature and an article in Times Higher Education , it is the largest academic social network in terms of active users.

Curriculum Vitae for R J Robbins

short personal version

Curriculum Vitae for R J Robbins

long standard version

RJR Picks from Around the Web (updated 11 MAY 2018 )