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18 Aug 2019 at 01:32
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Bibliography on: CRISPR-Cas


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 18 Aug 2019 at 01:32 Created: 


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: 2019-08-16

Keough KC, Lyalina S, Olvera MP, et al (2019)

AlleleAnalyzer: a tool for personalized and allele-specific sgRNA design.

Genome biology, 20(1):167 pii:10.1186/s13059-019-1783-3.

The CRISPR/Cas system is a highly specific genome editing tool capable of distinguishing alleles differing by even a single base pair. Target sites might carry genetic variations that are not distinguishable by sgRNA designing tools based on one reference genome. AlleleAnalyzer is an open-source software that incorporates single-nucleotide variants and short insertions and deletions to design sgRNAs for precisely editing 1 or multiple haplotypes of a sequenced genome, currently supporting 11 Cas proteins. It also leverages patterns of shared genetic variation to optimize sgRNA design for different human populations. AlleleAnalyzer is available at https://github.com/keoughkath/AlleleAnalyzer .

RevDate: 2019-08-16
CmpDate: 2019-08-16

Saeinasab M, Bahrami AR, González J, et al (2019)

SNHG15 is a bifunctional MYC-regulated noncoding locus encoding a lncRNA that promotes cell proliferation, invasion and drug resistance in colorectal cancer by interacting with AIF.

Journal of experimental & clinical cancer research : CR, 38(1):172 pii:10.1186/s13046-019-1169-0.

BACKGROUND: Thousands of long noncoding RNAs (lncRNAs) are aberrantly expressed in various types of cancers, however our understanding of their role in the disease is still very limited.

METHODS: We applied RNAseq analysis from patient-derived data with validation in independent cohort of patients. We followed these studies with gene regulation analysis as well as experimental dissection of the role of the identified lncRNA by multiple in vitro and in vivo methods.

RESULTS: We analyzed RNA-seq data from tumors of 456 CRC patients compared to normal samples, and identified SNHG15 as a potentially oncogenic lncRNA that encodes a snoRNA in one of its introns. The processed SNHG15 is overexpressed in CRC tumors and its expression is highly correlated with poor survival of patients. Interestingly, SNHG15 is more highly expressed in tumors with high levels of MYC expression, while MYC protein binds to two E-box motifs on SNHG15 sequence, indicating that SNHG15 transcription is directly regulated by the oncogene MYC. The depletion of SNHG15 by siRNA or CRISPR-Cas9 inhibits cell proliferation and invasion, decreases colony formation as well as the tumorigenic capacity of CRC cells, whereas its overexpression leads to opposite effects. Gene expression analysis performed upon SNHG15 inhibition showed changes in multiple relevant genes implicated in cancer progression, including MYC, NRAS, BAG3 or ERBB3. Several of these genes are functionally related to AIF, a protein that we found to specifically interact with SNHG15, suggesting that the SNHG15 acts, at least in part, by regulating the activity of AIF. Interestingly, ROS levels, which are directly regulated by AIF, show a significant reduction in SNHG15-depleted cells. Moreover, knockdown of SNHG15 increases the sensitiveness of the cells to 5-FU, while its overexpression renders them more resistant to the chemotherapeutic drug.

CONCLUSION: Altogether, these results describe an important role of SNHG15 in promoting colon cancer and mediating drug resistance, suggesting its potential as prognostic marker and target for RNA-based therapies.

RevDate: 2019-08-16
CmpDate: 2019-08-16

Dong Z, Wu S, Zhu C, et al (2019)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated kif15 mutations accelerate axonal outgrowth during neuronal development and regeneration in zebrafish.

Traffic (Copenhagen, Denmark), 20(1):71-81.

KIF15, the vertebrate kinesin-12, is best known as a mitotic motor protein, but continues to be expressed in neurons. Like KIF11 (the vertebrate kinesin-5), KIF15 interacts with microtubules in the axon to limit their sliding relative to one another. Unlike KIF11, KIF15 also regulates interactions between microtubules and actin filaments at sites of axonal branch formation and in growth cones. Our original work on these motors was done on cultured rat neurons, but we are now using zebrafish to extend these studies to an in vivo model. We previously studied kif15 in zebrafish by injecting splice-blocking morpholinos injected into embryos. Consistent with the cell culture work, these studies demonstrated that axons grow faster and longer when KIF15 levels are reduced. In the present study, we applied CRISPR/Cas9-based knockout technology to create kif15 mutants and labeled neurons with Tg(mnx1:GFP) transgene or transient expression of elavl3:EGFP-alpha tubulin. We then compared by live imaging the homozygotic, heterozygotic mutants to their wildtype siblings to ascertain the effects of depletion of kif15 during Caudal primary motor neuron and Rohon-Beard (R-B) sensory neuron development. The results showed, compared to the kif15 wildtype, the number of branches was reduced while axon outgrowth was accelerated in kif15 homozygotic and heterozygotic mutants. In R-B sensory neurons, after laser irradiation, injured axons with loss of kif15 displayed significantly greater regenerative velocity. Given these results and the fact that kif15 drugs are currently under development, we posit kif15 as a novel target for therapeutically augmenting regeneration of injured axons.

RevDate: 2019-08-16
CmpDate: 2019-08-16

Batool S, Argyropoulos KV, Azad R, et al (2019)

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells.

Biochimica et biophysica acta. General subjects, 1863(1):232-240.

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.

RevDate: 2019-08-16
CmpDate: 2019-08-16

Ivaldi MS, Diaz LF, Chakalova L, et al (2018)

Fetal γ-globin genes are regulated by the BGLT3 long noncoding RNA locus.

Blood, 132(18):1963-1973.

Long noncoding RNAs (lncRNAs) are increasingly being appreciated as participants in regulation of important cellular processes, including transcription. Because lncRNAs are highly cell type specific, they have the potential to contribute to the unique transcriptional repertoire of diverse cells, but underlying mechanisms are unclear. We studied BGLT3, an erythroid lncRNA encoded downstream of Aγ-globin (HBG1). BGLT3 and γ-globin genes are dynamically cotranscribed in erythroid cells in vivo. Deletion of BGLT3 using CRISPR/Cas9 editing shows that it specifically contributes to regulation of γ-globin genes. We used reduction or overexpression of the RNA and inhibition of transcription through the locus by CRISPRi to distinguish functions of the transcript vs the underlying sequence. Transcription of the BGLT3 locus is critical for looping between the γ-globin genes and BGLT3 sequences. In contrast, the BGLT3 transcript is dispensable for γ-globin/BGLT3 looping but interacts with the mediator complex on chromatin. Manipulation of the BGLT3 locus does not compromise γ-globin gene long-range looping interactions with the β-globin locus control region (LCR). These data reveal that BGLT3 regulates γ-globin transcription in a developmental stage-specific fashion together with the LCR by serving as a separate means to increase RNA Pol II density at the γ-globin promoters.

RevDate: 2019-08-16
CmpDate: 2019-08-16

Anonymous (2018)

CRISPR Causes Unexpected Genomic Damage.

Cancer discovery, 8(9):OF2.

A study in four cell lines concludes that CRISPR/Cas9 editing causes unanticipated genomic alterations, including large deletions, translocations, and insertions. Some of these changes could be the first hits that cause cells to eventually become neoplastic.

RevDate: 2019-08-16
CmpDate: 2019-08-16

van der Els S, James JK, Kleerebezem M, et al (2018)

Versatile Cas9-Driven Subpopulation Selection Toolbox for Lactococcus lactis.

Applied and environmental microbiology, 84(8):.

CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN-targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis, while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria.IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the context of potentially problematic prophages present in a strain. Moreover, Cas9 targeting of other mobile genetic elements enables the deciphering of their contribution to dairy fermentation processes and further establishment of their importance for product characteristics.

RevDate: 2019-08-14

Tang Z, Chen S, Chen A, et al (2019)

CasPDB: an integrated and annotated database for Cas proteins from bacteria and archaea.

Database : the journal of biological databases and curation, 2019:.

Clustered regularly interspaced short palindromic repeats (CRISPR) and associated proteins (Cas) constitute CRISPR-Cas systems, which are antiphage immune systems present in numerous bacterial and most archaeal species. In recent years, CRISPR-Cas systems have been developed into reliable and powerful genome editing tools. Nevertheless, finding similar or better tools from bacteria or archaea remains crucial. This requires the exploration of different CRISPR systems, identification and characterization new Cas proteins. Archives tailored for Cas proteins are urgently needed and necessitate the prediction and grouping of Cas proteins into an information center with all available experimental evidence. Here, we constructed Cas Protein Data Bank (CasPDB), an integrated and annotated online database for Cas proteins from bacteria and archaea. The CasPDB database contains 287 reviewed Cas proteins, 257 745 putative Cas proteins and 3593 Cas operons from 32 023 bacteria species and 1802 archaea species. The database can be freely browsed and searched. The CasPDB web interface also represents all the 3593 putative Cas operons and its components. Among these operons, 328 are members of the type II CRISPR-Cas system.

RevDate: 2019-08-14

Hullahalli K, Rodrigues M, Nguyen UT, et al (2019)

Erratum for Hullahalli et al., "An Attenuated CRISPR-Cas System in Enterococcus faecalis Permits DNA Acquisition".

mBio, 10(4): pii:mBio.01775-19.

RevDate: 2019-08-14

Gloag ES, Marshall CW, Snyder D, et al (2019)

Pseudomonas aeruginosa Interstrain Dynamics and Selection of Hyperbiofilm Mutants during a Chronic Infection.

mBio, 10(4): pii:mBio.01698-19.

Opportunistic pathogens establishing new infections experience strong selection to adapt, often favoring mutants that persist. Capturing this initial dynamic is critical for identifying the first adaptations that drive pathogenesis. Here we used a porcine full-thickness burn wound model of chronic infection to study the evolutionary dynamics of diverse Pseudomonas aeruginosa infections. Wounds were infected with a mixed community of six P. aeruginosa strains, including the model PA14 strain (PA14-1), and biopsies taken at 3, 14, and 28 days postinfection. Hyperbiofilm-forming rugose small-colony variants (RSCVs) were the earliest and predominant phenotypic variant. These variants were detected on day 3 and persisted, with the majority evolved from PA14-1. Whole-genome sequencing of PA14-1 RSCV isolates revealed driver mutations exclusively in the wsp pathway, conferring hyperbiofilm phenotypes. Several of the wsp mutant RSCVs also acquired CRISPR-Cas adaptive immunity to prophages isolated from the P. aeruginosa wound isolate (B23-2) that was also present in the inoculum. These observations emphasize the importance of interstrain dynamics and the role of lysogenic phages in the survival of an invading pathogen. Rather than being a side effect of chronicity, the rapid rise of RSCVs in wounds is evidence of positive selection on the Wsp chemosensory system to produce mutants with elevated biofilm formation capacity. We predict that RSCVs provide a level of phenotypic diversity to the infecting bacterial community and are common, early adaptations during infections. This would likely have significant consequences for clinical outcomes.IMPORTANCE Bacteria adapt to infections by evolving variants that are more fit and persistent. These recalcitrant variants are typically observed in chronic infections. However, it is unclear when and why these variants evolve. To address these questions, we used a porcine chronic wound model to study the evolutionary dynamics of Pseudomonas aeruginosa in a mixed-strain infection. We isolated hyperbiofilm variants that persisted early in the infection. Interstrain interactions were also observed, where adapted variants acquired CRISPR-mediated immunity to phages. We show that when initiating infection, P. aeruginosa experiences strong positive selection for hyperbiofilm phenotypes produced by mutants of a single chemosensory system, the Wsp pathway. We predict that hyperbiofilm variants are early adaptations to infection and that interstrain interactions may influence bacterial burden and infection outcomes.

RevDate: 2019-08-14
CmpDate: 2019-08-14

Niu XR, Yin SM, Chen X, et al (2019)

[Gene editing technology and its recent progress in disease therapy].

Yi chuan = Hereditas, 41(7):582-598.

Gene editing is a genetic manipulation technology which utilizes bacterial nucleases to accurately and efficiently modify DNA or RNA. Gene editing has broad applications in basic research, breeding, and drug screening, and it is gaining validity and applicability to the therapy of many diseases especially genetic-based disease. In this review, we summarize the development of gene editing technology, its different strategies and applications in the treatment of disease, and the research of gene editing therapy for genetic diseases (including base editor and epigenetic regulation) in the treatment of disorders and diseases of the blood system, liver, muscle and nervous system. Finally, we discuss the future development prospects of gene editing therapy.

RevDate: 2019-08-14
CmpDate: 2019-08-14

Wang J, Huang J, R Xu (2019)

[Seamless genome editing in Drosophila by combining CRISPR/Cas9 and piggyBac technologies].

Yi chuan = Hereditas, 41(5):422-429.

The type2 CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated protein 9) is an efficient RNA-guided genome-editing technique. Guided by sgRNA, the Cas9 endonuclease generates site-specific double-stranded breaks (DSB) at specific site, which is amenable to repair by homology-directed repair (HDR) to generate a designed knock-out or knock-in transgene. In combination with CRISPR/Cas9 and Cre/loxP or FLP/FRT system, efficient gene targeting can be achieved, and meanwhile screening markers introduced can be readily removed except a 34-base pair residual fragment. Thus, difficulties remain in accurate editing of the genome without introducing any extraneous sequences. In human induced pluripotent stem cells (iPSCs), a two-step strategy has been developed using CRISPR/Cas9 and the piggyBac system to establish a seamless genomic editing, in which CRISPR/Cas9 is initially used to introduce mutations along with screening markers by HDR, then the markers are precisely excised by piggyBac transposase. Using this strategy, we have successfully transformed the tyrosine to cysteine at position 21 within the 18th exon of the CG4894 gene in the Drosophila genome without introducing any extraneous sequence. Hence, this strategy provides more options for precise and seamless editing of the Drosophila genome.

RevDate: 2019-08-14
CmpDate: 2019-08-14

Bellin M (2018)

Crispr/Cas9 homologous recombination (HR).

Drug discovery today. Technologies, 28:1-2.

RevDate: 2019-08-14
CmpDate: 2019-08-14

Cromer MK, Vaidyanathan S, Ryan DE, et al (2018)

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells.

Molecular therapy : the journal of the American Society of Gene Therapy, 26(10):2431-2442.

Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34+ hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

RevDate: 2019-08-14
CmpDate: 2019-08-14

Zhan T, Rindtorff N, Betge J, et al (2019)

CRISPR/Cas9 for cancer research and therapy.

Seminars in cancer biology, 55:106-119.

CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer.

RevDate: 2019-08-13

Li QV, Rosen BP, D Huangfu (2019)

Decoding pluripotency: Genetic screens to interrogate the acquisition, maintenance, and exit of pluripotency.

Wiley interdisciplinary reviews. Systems biology and medicine [Epub ahead of print].

Pluripotent stem cells have the ability to unlimitedly self-renew and differentiate to any somatic cell lineage. A number of systems biology approaches have been used to define this pluripotent state. Complementary to systems level characterization, genetic screens offer a unique avenue to functionally interrogate the pluripotent state and identify the key players in pluripotency acquisition and maintenance, exit of pluripotency, and lineage differentiation. Here we review how genetic screens have helped us decode pluripotency regulation. We will summarize results from RNA interference (RNAi) based screens, discuss recent advances in CRISPR/Cas-based genetic perturbation methods, and how these advances have made it possible to more comprehensively interrogate pluripotency and differentiation through genetic screens. Such investigations will not only provide a better understanding of this unique developmental state, but may enhance our ability to use pluripotent stem cells as an experimental model to study human development and disease progression. Functional interrogation of pluripotency also provides a valuable roadmap for utilizing genetic perturbation to gain systems level understanding of additional cellular states, from later stages of development to pathological disease states. This article is categorized under: Developmental Biology > Stem Cell Biology and Regeneration Developmental Biology > Developmental Processes in Health and Disease Biological Mechanisms > Cell Fates.

RevDate: 2019-08-12

Hanewich-Hollatz MH, Chen Z, Hochrein LM, et al (2019)

Conditional Guide RNAs: Programmable Conditional Regulation of CRISPR/Cas Function in Bacterial and Mammalian Cells via Dynamic RNA Nanotechnology.

ACS central science, 5(7):1241-1249.

A guide RNA (gRNA) directs the function of a CRISPR protein effector to a target gene of choice, providing a versatile programmable platform for engineering diverse modes of synthetic regulation (edit, silence, induce, bind). However, the fact that gRNAs are constitutively active places limitations on the ability to confine gRNA activity to a desired location and time. To achieve programmable control over the scope of gRNA activity, here we apply principles from dynamic RNA nanotechnology to engineer conditional guide RNAs (cgRNAs) whose activity is dependent on the presence or absence of an RNA trigger. These cgRNAs are programmable at two levels, with the trigger-binding sequence controlling the scope of the effector activity and the target-binding sequence determining the subject of the effector activity. We demonstrate molecular mechanisms for both constitutively active cgRNAs that are conditionally inactivated by an RNA trigger (ON → OFF logic) and constitutively inactive cgRNAs that are conditionally activated by an RNA trigger (OFF → ON logic). For each mechanism, automated sequence design is performed using the reaction pathway designer within NUPACK to design an orthogonal library of three cgRNAs that respond to different RNA triggers. In E. coli expressing cgRNAs, triggers, and silencing dCas9 as the protein effector, we observe a median conditional response of ≈4-fold for an ON → OFF "terminator switch" mechanism, ≈15-fold for an ON → OFF "splinted switch" mechanism, and ≈3-fold for an OFF → ON "toehold switch" mechanism; the median crosstalk within each cgRNA/trigger library is <2%, ≈2%, and ≈20% for the three mechanisms. To test the portability of cgRNA mechanisms prototyped in bacteria to mammalian cells, as well as to test generalizability to different effector functions, we implemented the terminator switch in HEK 293T cells expressing inducing dCas9 as the protein effector, observing a median ON → OFF conditional response of ≈4-fold with median crosstalk of ≈30% for three orthogonal cgRNA/trigger pairs. By providing programmable control over both the scope and target of protein effector function, cgRNA regulators offer a promising platform for synthetic biology.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Huang Y, Xuan H, Yang C, et al (2019)

GmHsp90A2 is involved in soybean heat stress as a positive regulator.

Plant science : an international journal of experimental plant biology, 285:26-33.

Heat shock protein 90 s (Hsp90s), one of the most conserved and abundant molecular chaperones, is an essential component of the protective stress response. A previous study reported at least 12 genes in the GmHsp90s family in soybean and that GmHsp90A2 overexpression enhanced thermotolerance in Arabidopsis thaliana. Here, we investigate the roles of GmHsp90A2 in soybean by utilizing stable transgenic soybean lines overexpressing GmHsp90A2 and mutant lines generated by the CRISPR/Cas9 system. The results showed that compared with wild-type plants (WT) and empty vector control plants (VC), T3 transgenic soybean plants overexpressing GmHsp90A2 exhibited increased tolerance to heat stress through higher chlorophyll and lower malondialdehyde (MDA) contents in plants. Conversely, reduced chlorophyll and increased MDA contents in T2 homozygous GmHsp90A2-knockout mutants indicated decreased tolerance to heat stress. GmHsp90A2 was found to interact with GmHsp90A1 in yeast two-hybrid assays. Furthermore, subcellular localization analyses revealed that GmHsp90A2 was localized to the cytoplasm and cell membrane; as shown by bimolecular fluorescence complementation (BiFC) assays, GmHsp90A2 interacted with GmHsp90A1 in the nucleus and cytoplasm and cell membrane. Hence, we conclude that GmHsp90A1 is able to bind to GmHsp90A2 to form a complex and that this complex enters the nucleus. In summary, GmHsp90A2 might respond to heat stress and positively regulate thermotolerance by interacting with GmHsp90A1.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Albrechtsen R, Wewer Albrechtsen NJ, Gnosa S, et al (2019)

Identification of ADAM12 as a Novel Basigin Sheddase.

International journal of molecular sciences, 20(8): pii:ijms20081957.

The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Zhai Y, Cai S, Hu L, et al (2019)

CRISPR/Cas9-mediated genome editing reveals differences in the contribution of INDEHISCENT homologues to pod shatter resistance in Brassica napus L.

TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik, 132(7):2111-2123.

The INDEHISCENT (IND) and ALCATRAZ (ALC) gene homologues have been reported to be essential for dehiscence of fruits in Brassica species. But their functions for pod shatter resistance in Brassica napus, an important oil crops, are not well understood. Here, we assessed the functions of these two genes in rapeseed using CRISPR/Cas9 technology. The induced mutations were stably transmitted to successive generations, and a variety of homozygous mutants with loss-of-function alleles of the target genes were obtained for phenotyping. The results showed that the function of BnIND gene is essential for pod shatter and highly conserved in Brassica species, whereas the BnALC gene appears to have limited potential for rapeseed shatter resistance. The homoeologous copies of the BnIND gene have partially redundant roles in rapeseed pod shatter, with BnA03.IND exhibiting higher contributions than BnC03.IND. Analysis of data obtained from the gene expression and sequence variations of gene copies revealed that cis-regulatory divergences alter gene expression and underlie the functional differentiation of BnIND homologues. Collectively, our results generate valuable resources for rapeseed breeding programs, and more importantly provide a strategy to improve polyploid crops.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Cyranoski D (2019)

The CRISPR-baby scandal: what's next for human gene-editing.

Nature, 566(7745):440-442.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Kuil LE, Oosterhof N, Geurts SN, et al (2019)

Reverse genetic screen reveals that Il34 facilitates yolk sac macrophage distribution and seeding of the brain.

Disease models & mechanisms, 12(3): pii:dmm.037762.

Microglia are brain-resident macrophages, which have specialized functions important in brain development and in disease. They colonize the brain in early embryonic stages, but few factors that drive the migration of yolk sac macrophages (YSMs) into the embryonic brain, or regulate their acquisition of specialized properties, are currently known. Here, we present a CRISPR/Cas9-based in vivo reverse genetic screening pipeline to identify new microglia regulators using zebrafish. Zebrafish larvae are particularly suitable due to their external development, transparency and conserved microglia features. We targeted putative microglia regulators, by Cas9/gRNA complex injections, followed by Neutral-Red-based visualization of microglia. Microglia were quantified automatically in 3-day-old larvae using a software tool we called SpotNGlia. We identified that loss of zebrafish colony-stimulating factor 1 receptor (Csf1r) ligand, Il34, caused reduced microglia numbers. Previous studies on the role of IL34 in microglia development in vivo were ambiguous. Our data, and a concurrent paper, show that, in zebrafish, il34 is required during the earliest seeding of the brain by microglia. Our data also indicate that Il34 is required for YSM distribution to other organs. Disruption of the other Csf1r ligand, Csf1, did not reduce microglia numbers in mutants, whereas overexpression increased the number of microglia. This shows that Csf1 can influence microglia numbers, but might not be essential for the early seeding of the brain. In all, we identified il34 as a modifier of microglia colonization, by affecting distribution of YSMs to target organs, validating our reverse genetic screening pipeline in zebrafish.This article has an associated First Person interview with the joint first authors of the paper.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Hayashi A, K Tanaka (2019)

Short-Homology-Mediated CRISPR/Cas9-Based Method for Genome Editing in Fission Yeast.

G3 (Bethesda, Md.), 9(4):1153-1163 pii:g3.118.200976.

The CRISPR/Cas9 system enables the editing of genomes of numerous organisms through the induction of the double-strand breaks (DSB) at specific chromosomal targets. We improved the CRISPR/Cas9 system to ease the direct introduction of a point mutation or a tagging sequence into the chromosome by combining it with the noncanonical homology-directed DNA repair (HDR) based genome editing in fission yeast. We constructed convenient cloning vectors, which possessed a guide RNA (gRNA) expression module, or the humanized Streptococcus pyogenes Cas9 gene that is expressed under the control of an inducible promoter to avoid the needless expression, or both a gRNA and Cas9 gene. Using this system, we attempted the short-homology-mediated genome editing and found that the HDR pathway provides high-frequency genome editing at target loci without the need of a long donor DNA. Using short oligonucleotides, we successfully introduced point mutations into two target genes at high frequency. We also precisely integrated the sequences for epitope and GFP tagging using donor DNA possessing short homology into the target loci, which enabled us to obtain cells expressing N-terminally tagged fusion proteins. This system could expedite genome editing in fission yeast, and could be applicable to other organisms.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Hollerer I, Barker JC, Jorgensen V, et al (2019)

Evidence for an Integrated Gene Repression Mechanism Based on mRNA Isoform Toggling in Human Cells.

G3 (Bethesda, Md.), 9(4):1045-1053 pii:g3.118.200802.

We recently described an unconventional mode of gene regulation in budding yeast by which transcriptional and translational interference collaborate to down-regulate protein expression. Developmentally timed transcriptional interference inhibited production of a well translated mRNA isoform and resulted in the production of an mRNA isoform containing inhibitory upstream open reading frames (uORFs) that prevented translation of the main ORF. Transcriptional interference and uORF-based translational repression are established mechanisms outside of yeast, but whether this type of integrated regulation was conserved was unknown. Here we find that, indeed, a similar type of regulation occurs at the locus for the human oncogene MDM2 We observe evidence of transcriptional interference between the two MDM2 promoters, which produce a poorly translated distal promoter-derived uORF-containing mRNA isoform and a well-translated proximal promoter-derived transcript. Down-regulation of distal promoter activity markedly up-regulates proximal promoter-driven expression and results in local reduction of histone H3K36 trimethylation. Moreover, we observe that this transcript toggling between the two MDM2 isoforms naturally occurs during human embryonic stem cell differentiation programs.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Voutev R, RS Mann (2019)

TP901-1 Phage Recombinase Facilitates Genome Engineering in Drosophila melanogaster.

G3 (Bethesda, Md.), 9(4):983-986 pii:g3.119.0002.

Molecular biology techniques have a large impact on biomedical research and the availability of diverse tools to perform genome manipulations advances the ease of executing complicated genetic research. Here, we introduce in the fruit fly another such tool by harnessing the phage recombinase TP901-1 to perform site-directed recombination that leads to recombinase-mediated cassette exchange (RMCE). The TP901-1 system complements already existing recombination systems and enhances genome engineering in the fruit fly and other organisms.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Liu H, Li DM, Zhu LY, et al (2019)

[Research on the knockout of LMNA gene by CRISPR/Cas9 system in human cell lines].

Yi chuan = Hereditas, 41(1):66-75.

The LMNA gene encodes the nuclear Lamin A and Lamin C proteins, and is related to nuclear membrane organization, genome stability and cell differentiation. Abnormal expression of LMNA is ubiquitous in human tumors, and its mutation leads to various forms of laminopathies, including Emery-Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), and Hutchinson-Gliford progeria syndrome (HGPS). To further determine the functions of the LMNA gene in cellular physiology, the present study used the CRISPR/Cas9 technique to edit the LMNA gene of 293T and HepG2 cells in vitro, which resulted in two stable LMNA gene knockout (LMNA KO) cell lines. Compared to the respective wild type cells, the LMNA KO cell lines showed decrease in proliferation ability, increase in apoptosis, alteration in cellular morphology and uneven structures in the nucleus membrane. In this study, we report for the first time the results on the construction of LMNA KO immortalized cell lines and characterization of their morphological changes, thereby laying the foundation for the further studies of the LMNA gene functions and pathogenic mutations.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Durand GA, Raoult D, G Dubourg (2019)

Antibiotic discovery: history, methods and perspectives.

International journal of antimicrobial agents, 53(4):371-382.

Antimicrobial resistance is considered a major public-health issue. Policies recommended by the World Health Organization (WHO) include research on new antibiotics. No new class has been discovered since daptomycin and linezolid in the 1980s, and only optimisation or combination of already known compounds has been recently commercialised. Antibiotics are natural products of soil-living organisms. Actinobacteria and fungi are the source of approximately two-thirds of the antimicrobial agents currently used in human medicine; they were mainly discovered during the golden age of antibiotic discovery. This era declined after the 1970s owing to the difficulty of cultivating fastidious bacterial species under laboratory conditions. Various strategies, such as rational drug design, to date have not led to the discovery of new antimicrobial agents. However, new promising approaches, e.g. genome mining or CRISPR-Cas9, are now being developed. The recent rebirth of culture methods from complex samples has, as a matter of fact, permitted the discovery of teixobactin from a new species isolated from soil. Recently, many biosynthetic gene clusters were identified from human-associated microbiota, especially from the gut and oral cavity. For example, the antimicrobial lugdunin was recently discovered in the oral cavity. The repertoire of human gut microbiota has recently substantially increased, with the discovery of hundreds of new species. Exploration of the repertoire of prokaryotes associated with humans using genome mining or newer culture approaches could be promising strategies for discovering new classes of antibiotics.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Lavender P, Kelly A, Hendy E, et al (2018)

CRISPR-based reagents to study the influence of the epigenome on gene expression.

Clinical and experimental immunology, 194(1):9-16.

The use of epigenome editing is set to expand our knowledge of how epigenetic landscapes facilitate gene expression capacity within a given cell. As epigenetic landscape profiling in health and disease becomes more commonplace, so does the requirement to assess the functional impact that particular regulatory domains and DNA methylation profiles have upon gene expression capacity. That functional assessment is particularly pertinent when analysing epigenomes in disease states where the reversible nature of histone and DNA modification might yield plausible therapeutic targets. In this review we discuss first the nature of the epigenetic landscape, secondly the types of factors that deposit and erase the various modifications, consider how modifications transduce their signals, and lastly address current tools for experimental epigenome editing with particular emphasis on the immune system.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Dastidar S, Ardui S, Singh K, et al (2018)

Efficient CRISPR/Cas9-mediated editing of trinucleotide repeat expansion in myotonic dystrophy patient-derived iPS and myogenic cells.

Nucleic acids research, 46(16):8275-8298.

CRISPR/Cas9 is an attractive platform to potentially correct dominant genetic diseases by gene editing with unprecedented precision. In the current proof-of-principle study, we explored the use of CRISPR/Cas9 for gene-editing in myotonic dystrophy type-1 (DM1), an autosomal-dominant muscle disorder, by excising the CTG-repeat expansion in the 3'-untranslated-region (UTR) of the human myotonic dystrophy protein kinase (DMPK) gene in DM1 patient-specific induced pluripotent stem cells (DM1-iPSC), DM1-iPSC-derived myogenic cells and DM1 patient-specific myoblasts. To eliminate the pathogenic gain-of-function mutant DMPK transcript, we designed a dual guide RNA based strategy that excises the CTG-repeat expansion with high efficiency, as confirmed by Southern blot and single molecule real-time (SMRT) sequencing. Correction efficiencies up to 90% could be attained in DM1-iPSC as confirmed at the clonal level, following ribonucleoprotein (RNP) transfection of CRISPR/Cas9 components without the need for selective enrichment. Expanded CTG repeat excision resulted in the disappearance of ribonuclear foci, a quintessential cellular phenotype of DM1, in the corrected DM1-iPSC, DM1-iPSC-derived myogenic cells and DM1 myoblasts. Consequently, the normal intracellular localization of the muscleblind-like splicing regulator 1 (MBNL1) was restored, resulting in the normalization of splicing pattern of SERCA1. This study validates the use of CRISPR/Cas9 for gene editing of repeat expansions.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Takahashi T, Nakano Y, Onomoto K, et al (2018)

LGP2 virus sensor regulates gene expression network mediated by TRBP-bound microRNAs.

Nucleic acids research, 46(17):9134-9147.

Here we show that laboratory of genetics and physiology 2 (LGP2) virus sensor protein regulates gene expression network of endogenous genes mediated by TAR-RNA binding protein (TRBP)-bound microRNAs (miRNAs). TRBP is an enhancer of RNA silencing, and functions to recruit precursor-miRNAs (pre-miRNAs) to Dicer that processes pre-miRNA into mature miRNA. Viral infection activates the antiviral innate immune response in mammalian cells. Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including RIG-I, melanoma-differentiation-associated gene 5 (MDA5), and LGP2, function as cytoplasmic virus sensor proteins during viral infection. RIG-I and MDA5 can distinguish between different types of RNA viruses to produce antiviral cytokines, including type I interferon. However, the role of LGP2 is controversial. We found that LGP2 bound to the double-stranded RNA binding sites of TRBP, resulting in inhibition of pre-miRNA binding and recruitment by TRBP. Furthermore, although it is unclear whether TRBP binds to specific pre-miRNA, we found that TRBP bound to particular pre-miRNAs with common structural characteristics. Thus, LGP2 represses specific miRNA activities by interacting with TRBP, resulting in selective regulation of target genes. Our findings show that a novel function of LGP2 is to modulate RNA silencing, indicating the crosstalk between RNA silencing and RLR signaling in mammalian cells.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Sturm Á, Saskoi É, Tibor K, et al (2018)

Highly efficient RNAi and Cas9-based auto-cloning systems for C. elegans research.

Nucleic acids research, 46(17):e105.

RNA interference (RNAi) technology used for the functional analysis of Caenorhabditis elegans genes frequently leads to phenotypes with low penetrance or even proves completely ineffective. The methods previously developed to solve this problem were built on mutant genetic backgrounds, such as those defective for rrf-3, in which endogenous RNAi pathways are overexpressed. These mutations, however, interferes with many other genetic pathways so that the detected phenotype cannot always be clearly linked to the RNAi-exposed gene. In addition, using RNAi-overexpressing mutant backgrounds requires time-consuming genetic crossing. Here, we present an improved RNAi vector that produces specific double-stranded RNA species only, and thereby significantly stronger phenotypes than the standard gene knockdown vector. The further advantage of the new RNAi vector is that the detected phenotype can be specifically linked to the gene silenced. We also created a new all-in-one C. elegans Cas9 vector whose spacer sequence is much easier to replace. Both new vectors include a novel CRISPR/Cas9-based auto-cloning vector system rendering needless the use of restriction and ligase enzymes in generating DNA constructs. This novel, efficient RNAi and auto-cloning Cas9 systems can be easily adapted to any other genetic model.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Tasan I, Sustackova G, Zhang L, et al (2018)

CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci.

Nucleic acids research, 46(17):e100.

Nuclear organization has an important role in determining genome function; however, it is not clear how spatiotemporal organization of the genome relates to functionality. To elucidate this relationship, a method for tracking any locus of interest is desirable. Recently clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) or transcription activator-like effectors were adapted for imaging endogenous loci; however, they are mostly limited to visualization of repetitive regions. Here, we report an efficient and scalable method named SHACKTeR (Short Homology and CRISPR/Cas9-mediated Knock-in of a TetO Repeat) for live cell imaging of specific chromosomal regions without the need for a pre-existing repetitive sequence. SHACKTeR requires only two modifications to the genome: CRISPR/Cas9-mediated knock-in of an optimized TetO repeat and its visualization by TetR-EGFP expression. Our simplified knock-in protocol, utilizing short homology arms integrated by polymerase chain reaction, was successful at labeling 10 different loci in HCT116 cells. We also showed the feasibility of knock-in into lamina-associated, heterochromatin regions, demonstrating that these regions prefer non-homologous end joining for knock-in. Using SHACKTeR, we were able to observe DNA replication at a specific locus by long-term live cell imaging. We anticipate the general applicability and scalability of our method will enhance causative analyses between gene function and compartmentalization in a high-throughput manner.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Vasquez JJ, Wedel C, Cosentino RO, et al (2018)

Exploiting CRISPR-Cas9 technology to investigate individual histone modifications.

Nucleic acids research, 46(18):e106.

Despite their importance for most DNA-templated processes, the function of individual histone modifications has remained largely unknown because in vivo mutational analyses are lacking. The reason for this is that histone genes are encoded by multigene families and that tools to simultaneously edit multiple genomic loci with high efficiency are only now becoming available. To overcome these challenges, we have taken advantage of the power of CRISPR-Cas9 for precise genome editing and of the fact that most DNA repair in the protozoan parasite Trypanosoma brucei occurs via homologous recombination. By establishing an episome-based CRISPR-Cas9 system for T. brucei, we have edited wild type cells without inserting selectable markers, inserted a GFP tag between an ORF and its 3'UTR, deleted both alleles of a gene in a single transfection, and performed precise editing of genes that exist in multicopy arrays, replacing histone H4K4 with H4R4 in the absence of detectable off-target effects. The newly established genome editing toolbox allows for the generation of precise mutants without needing to change other regions of the genome, opening up opportunities to study the role of individual histone modifications, catalytic sites of enzymes or the regulatory potential of UTRs in their endogenous environments.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Prykhozhij SV, Fuller C, Steele SL, et al (2018)

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

Nucleic acids research, 46(17):e102.

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Couvin D, Bernheim A, Toffano-Nioche C, et al (2018)

CRISPRCasFinder, an update of CRISRFinder, includes a portable version, enhanced performance and integrates search for Cas proteins.

Nucleic acids research, 46(W1):W246-W251.

CRISPR (clustered regularly interspaced short palindromic repeats) arrays and their associated (Cas) proteins confer bacteria and archaea adaptive immunity against exogenous mobile genetic elements, such as phages or plasmids. CRISPRCasFinder allows the identification of both CRISPR arrays and Cas proteins. The program includes: (i) an improved CRISPR array detection tool facilitating expert validation based on a rating system, (ii) prediction of CRISPR orientation and (iii) a Cas protein detection and typing tool updated to match the latest classification scheme of these systems. CRISPRCasFinder can either be used online or as a standalone tool compatible with Linux operating system. All third-party software packages employed by the program are freely available. CRISPRCasFinder is available at https://crisprcas.i2bc.paris-saclay.fr.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Concordet JP, M Haeussler (2018)

CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens.

Nucleic acids research, 46(W1):W242-W245.

CRISPOR.org is a web tool for genome editing experiments with the CRISPR-Cas9 system. It finds guide RNAs in an input sequence and ranks them according to different scores that evaluate potential off-targets in the genome of interest and predict on-target activity. The list of genomes is continuously expanded, with more 150 genomes added in the last two years. CRISPOR tries to provide a comprehensive solution from selection, cloning and expression of guide RNA as well as providing primers needed for testing guide activity and potential off-targets. Recent developments include batch design for genome-wide CRISPR and saturation screens, creating custom oligonucleotides for guide cloning and the design of next generation sequencing primers to test for off-target mutations. CRISPOR is available from http://crispor.org, including the full source code of the website and a stand-alone, command-line version.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Lieberman J (2018)

Tapping the RNA world for therapeutics.

Nature structural & molecular biology, 25(5):357-364.

A recent revolution in RNA biology has led to the identification of new RNA classes with unanticipated functions, new types of RNA modifications, an unexpected multiplicity of alternative transcripts and widespread transcription of extragenic regions. This development in basic RNA biology has spawned a corresponding revolution in RNA-based strategies to generate new types of therapeutics. Here, I review RNA-based drug design and discuss barriers to broader applications and possible ways to overcome them. Because they target nucleic acids rather than proteins, RNA-based drugs promise to greatly extend the domain of 'druggable' targets beyond what can be achieved with small molecules and biologics.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Joseph B, Kondo S, EC Lai (2018)

Short cryptic exons mediate recursive splicing in Drosophila.

Nature structural & molecular biology, 25(5):365-371.

Many long Drosophila introns are processed by an unusual recursive strategy. The presence of ~200 adjacent splice acceptor and splice donor sites, termed ratchet points (RPs), were inferred to reflect 'zero-nucleotide exons', whose sequential processing subdivides removal of long host introns. We used CRISPR-Cas9 to disrupt several intronic RPs in Drosophila melanogaster, some of which recapitulated characteristic loss-of-function phenotypes. Unexpectedly, selective disruption of RP splice donors revealed constitutive retention of unannotated short exons. Assays using functional minigenes confirm that unannotated cryptic splice donor sites are critical for recognition of intronic RPs, demonstrating that recursive splicing involves the recognition of cryptic RP exons. This appears to be a general mechanism, because canonical, conserved splice donors are specifically enriched in a 40-80-nt window downstream of known and newly annotated intronic RPs and exhibit similar properties to a broadly expanded class of expressed RP exons. Overall, these studies unify the mechanism of Drosophila recursive splicing with that in mammals.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Tang L, Zeng Y, Zhou X, et al (2018)

Highly efficient ssODN-mediated homology-directed repair of DSBs generated by CRISPR/Cas9 in human 3PN zygotes.

Molecular reproduction and development, 85(6):461-463.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Cheng L, Tang Y, Chen X, et al (2018)

Deletion of MBD2 inhibits proliferation of chronic myeloid leukaemia blast phase cells.

Cancer biology & therapy, 19(8):676-686.

Aberrant methylation of tumour suppressor genes is associated with the progression to a blast crisis in chronic myeloid leukaemia (CML). Methyl-CpG-binding domain protein 2 (MBD2) has been studied as a "reader" of DNA methylation in many cancers, but its role in CML is unclear. We constructed cell models of a homozygous deletion mutation of MBD2 using gene-editing technology in K562 cells and BV173 cells. Here, we demonstrated that the deletion of MBD2 inhibited cell proliferation capacity in vitro. MBD2 deletion also significantly inhibited K562 cell proliferation in a xenograft tumour model in vivo. Additionally, the JAK2/STAT3 signalling pathway, which is abnormally active in CML, was inhibited by MBD2 deletion, and MBD2 deletion could up-regulate the expression of SHP1. In conclusion, our findings suggest that MBD2 is a candidate therapeutic strategy for the CML blast phase.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Baylis F (2018)

Counterpoint: The Potential Harms of Human Gene Editing Using CRISPR-Cas9.

Clinical chemistry, 64(3):489-491.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Katsanis N (2018)

Point: Treating Human Genetic Disease One Base Pair at a Time: The Benefits of Gene Editing.

Clinical chemistry, 64(3):486-488.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Schinazi RF, Ehteshami M, Bassit L, et al (2018)

Towards HBV curative therapies.

Liver international : official journal of the International Association for the Study of the Liver, 38 Suppl 1:102-114.

Tremendous progress has been made over the last 2 decades to discover and develop approaches to control hepatitis B virus (HBV) infections and to prevent the development of hepatocellular carcinoma using various interferons and small molecules as antiviral agents. However, none of these agents have significant impact on eliminating HBV from infected cells. Currently the emphasis is on silencing or eliminating cccDNA, which could lead to a cure for HBV. Various approaches are being developed including the development of capsid effectors, CRISPR/Cas9, TALENS, siRNA, entry and secretion inhibitors, as well as immunological approaches. It is very likely that a combination of these modalities will need to be employed to successfully eliminate HBV or prevent virus rebound on discontinuation of therapy. In the next 5 years clinical data will emerge which will provide insight on the safety and feasibility of these approaches and if they can be applied to eradicate HBV infections globally. In this review, we summarize current treatments and we highlight and examine recent therapeutic strategies that are currently being evaluated at the preclinical and clinical stage.

RevDate: 2019-08-13
CmpDate: 2019-08-13

Vilarino M, Rashid ST, Suchy FP, et al (2017)

CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep.

Scientific reports, 7(1):17472.

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1-/- fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

RevDate: 2019-08-12
CmpDate: 2019-08-12

Dampier W, Sullivan NT, Chung CH, et al (2017)

Designing broad-spectrum anti-HIV-1 gRNAs to target patient-derived variants.

Scientific reports, 7(1):14413.

Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9), including specific guide RNAs (gRNAs), can excise integrated human immunodeficiency virus type 1 (HIV-1) provirus from host chromosomes. To date, anti-HIV-1 gRNAs have been designed to account for off-target activity, however, they seldom account for genetic variation in the HIV-1 genome within and between patients, which will be crucial for therapeutic application of this technology. This analysis tests the ability of published anti-HIV-1 gRNAs to cleave publicly available patient-derived HIV-1 sequences to inform gRNA design and provides basic computational tools to researchers in the field.

RevDate: 2019-07-29
CmpDate: 2019-07-29

Bashir S, R Kühn (2017)

Enhanced precision and efficiency.

Nature biomedical engineering, 1(11):856-857.

RevDate: 2019-07-29
CmpDate: 2019-07-29

Anonymous (2017)

The expanding toolbox for genome engineering.

Nature biomedical engineering, 1(11):853.

RevDate: 2019-08-11

Hsu CT, Cheng YJ, Yuan YH, et al (2019)

Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco.

Plant molecular biology pii:10.1007/s11103-019-00907-w [Epub ahead of print].

KEY MESSAGE: Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants. Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.

RevDate: 2019-08-10

Liu X, Li G, Zhou X, et al (2019)

Improving Editing Efficiency for the Sequences with NGH PAM Using xCas9-Derived Base Editors.

Molecular therapy. Nucleic acids, 17:626-635 pii:S2162-2531(19)30189-1 [Epub ahead of print].

The development of CRISPR/Cas9-mediated base editors (BEs) provided a versatile tool for precise genome editing. The recently developed xCas9-derived base editors (xBEs) that recognize the NG PAM substantially expand the targeting scope in the genome, while their editing efficiency needs to be improved. Here, we described an improved version of xBEs by fusing the BPNLS and Gam to the N terminus of xBEs (BPNLS-Gam-xBE3 and BPNLS-xABE), and this version of base editor displayed higher targeting efficiency for the majority of detected sites. By using this improved version of xBEs, we successfully created and corrected pathogenic mutations at genomic sites with the NGN protospacer-adjacent motif in human cells. Lastly, we used BPNLS-Gam-xBE3 to model pathogenic mutations in discarded human tripronuclear (3PN) zygotes, and no obvious off-targets and indels were detected. Taken together, the data in our study offer an efficient tool for precise genome editing and, thus, an enriched base editing toolkit.

RevDate: 2019-08-10

Maikova A, Kreis V, Boutserin A, et al (2019)

Using endogenous CRISPR-Cas system for genome editing in the human pathogen Clostridium difficile.

Applied and environmental microbiology pii:AEM.01416-19 [Epub ahead of print].

Human enteropathogen Clostridium difficile constitutes a key public health issue in industrialized countries. Many aspects of C. difficile pathophysiology and adaptation inside the host remain poorly understood. We have recently reported that this bacterium possesses an active CRISPR-Cas system of subtype I-B for defence against phages and other mobile genetic elements that could contribute to its success during infection. In this paper, we demonstrate that redirecting this endogenous CRISPR-Cas system towards autoimmunity allows efficient genome editing in C. difficile We provide detailed description of this newly developed approach and show, as a proof of principle, its efficient application for deletion of a specific gene in reference 630Δerm and in epidemic R20291 C. difficile strains. The new method expands the arsenal of the currently limiting set of gene engineering tools available for investigation of C. difficile and may serve as the basis for new strategies to control C. difficile infections.ImportanceClostridium difficile represents today a real danger for human and animal health. It is the leading cause of diarrhoea associated with healthcare in adults in industrialized countries. The incidence of these infections continues to increase and this trend is accentuated by the general aging of the population. Many questions remain unanswered on the mechanisms contributing to C. difficile success inside the host. The set of genetic tools available for this pathogen is limited and new developments are badly needed. C. difficile has developed efficient defence systems that are directed against foreign DNA and could contribute to its survival in phage-rich gut communities. We show how one of such defence systems, named CRISPR-Cas, can be hijacked for C. difficile genome editing. Our results also show a great potential of CRISPR-Cas system for development of new therapeutic strategies against C. difficile infections.

RevDate: 2019-08-10

Kruse T, Ratnadevi CM, Erikstad HA, et al (2019)

Complete genome sequence analysis of the thermoacidophilic verrucomicrobial methanotroph "Candidatus Methylacidiphilum kamchatkense" strain Kam1 and comparison with its closest relatives.

BMC genomics, 20(1):642 pii:10.1186/s12864-019-5995-4.

BACKGROUND: The candidate genus "Methylacidiphilum" comprises thermoacidophilic aerobic methane oxidizers belonging to the Verrucomicrobia phylum. These are the first described non-proteobacterial aerobic methane oxidizers. The genes pmoCAB, encoding the particulate methane monooxygenase do not originate from horizontal gene transfer from proteobacteria. Instead, the "Ca. Methylacidiphilum" and the sister genus "Ca. Methylacidimicrobium" represent a novel and hitherto understudied evolutionary lineage of aerobic methane oxidizers. Obtaining and comparing the full genome sequences is an important step towards understanding the evolution and physiology of this novel group of organisms.

RESULTS: Here we present the closed genome of "Ca. Methylacidiphilum kamchatkense" strain Kam1 and a comparison with the genomes of its two closest relatives "Ca. Methylacidiphilum fumariolicum" strain SolV and "Ca. Methylacidiphilum infernorum" strain V4. The genome consists of a single 2,2 Mbp chromosome with 2119 predicted protein coding sequences. Genome analysis showed that the majority of the genes connected with metabolic traits described for one member of "Ca. Methylacidiphilum" is conserved between all three genomes. All three strains encode class I CRISPR-cas systems. The average nucleotide identity between "Ca. M. kamchatkense" strain Kam1 and strains SolV and V4 is ≤95% showing that they should be regarded as separate species. Whole genome comparison revealed a high degree of synteny between the genomes of strains Kam1 and SolV. In contrast, comparison of the genomes of strains Kam1 and V4 revealed a number of rearrangements. There are large differences in the numbers of transposable elements found in the genomes of the three strains with 12, 37 and 80 transposable elements in the genomes of strains Kam1, V4 and SolV respectively. Genomic rearrangements and the activity of transposable elements explain much of the genomic differences between strains. For example, a type 1h uptake hydrogenase is conserved between strains Kam1 and SolV but seems to have been lost from strain V4 due to genomic rearrangements.

CONCLUSIONS: Comparing three closed genomes of "Ca. Methylacidiphilum" spp. has given new insights into the evolution of these organisms and revealed large differences in numbers of transposable elements between strains, the activity of these explains much of the genomic differences between strains.

RevDate: 2019-08-09

Knott GJ, Cress BF, Liu JJ, et al (2019)

Structural basis for AcrVA4 inhibition of specific CRISPR-Cas12a.

eLife, 8: pii:49110 [Epub ahead of print].

CRISPR-Cas systems provide bacteria and archaea with programmable immunity against mobile genetic elements. Evolutionary pressure by CRISPR-Cas has driven bacteriophage to evolve small protein inhibitors, anti-CRISPRs (Acrs), that block Cas enzyme function by wide-ranging mechanisms. We show here that the inhibitor AcrVA4 uses a previously undescribed strategy to recognize the L. bacterium Cas12a (LbCas12a) pre-crRNA processing nuclease, forming a Cas12a dimer, and allosterically inhibiting DNA binding. The A. species Cas12a (AsCas12a) enzyme, widely used for genome editing applications, contains an ancestral helical bundle that blocks AcrVA4 binding and allows it to escape anti-CRISPR recognition. Using biochemical, microbiological, and human cell editing experiments, we show that Cas12a orthologs can be rendered either sensitive or resistant to AcrVA4 through rational structural engineering informed by evolution. Together, these findings explain a new mode of CRISPR-Cas inhibition and illustrate how structural variability in Cas effectors can drive opportunistic co-evolution of inhibitors by bacteriophage.

RevDate: 2019-08-09
CmpDate: 2019-08-09

Wang C, Cheng W, Yu Q, et al (2018)

Toxoplasma Chinese 1 Strain of WH3Δrop16I/III /gra15II Genetic Background Contributes to Abnormal Pregnant Outcomes in Murine Model.

Frontiers in immunology, 9:1222.

Toxoplasma gondii infection evokes a strong Th1-type response with interleukin (IL)-12 and interferon (IFN)-γ secretion. Recent studies suggest that the infection of pregnant mice with T. gondii may lead to adverse pregnancy results caused by subversion of physiological immune tolerance at maternofetal interface rather than direct invasion of the parasite. Genotype-associated dense granule protein GRA15II tends to induce classically activated macrophage (M1) differentiation and subsequently activating NK, Th1, and Th17 cells whereas rhoptry protein ROP16I/III drives macrophages to alternatively activated macrophage (M2) polarization and elicits Th2 immune response. Unlike the archetypal strains of types I, II, and III, type Chinese 1 strains possess both GRA15II and ROP16I/III, suggesting a distinct pathogenesis of Toxoplasma-involved adverse pregnancies. We constructed T. gondii type Chinese 1 strain of WH3Δrop16 based on CRISPR/Cas9 technology to explore the ROP16I/III-deficient/GRA15II-dominant parasites in induction of trophoblast apoptosis in vitro and abnormal pregnant outcomes of mice in vivo. Our study showed that Toxoplasma WH3Δrop16 remarkably induced apoptosis of trophoblasts. C57BL/6 pregnant mice injected with the tachyzoites of WH3Δrop16 presented increased absorptivity of fetuses in comparison with the mice infected with WH3 wild type (WH3 WT) parasites although no remarkable difference of virulence to mice was seen between the two strains. Additionally, the mice inoculated with WH3Δrop16 tachyzoites exhibited a notable expression of both IL-17A and IFN-γ, while the percentage of CD4+CD25+FoxP3 [T regulatory cells (Tregs)] were diminished in splenocytes and placenta tissues compared to those infected with WH3 WT parasites. Accordingly, expressions of IL-4, IL-10, and transforming growth factor beta 1, the pivotal cytokines of Th2 and Tregs response, were significantly dampened whereas IFN-γ and IL-12 expressions were upregulated in WH3Δrop16-infected mice, which gave rise to more prominent outcomes of abnormal pregnancies. Our results indicated that the WH3Δrop16 parasites with gra15II background of T. gondii type Chinese 1 strains may cause miscarriage and stillbirth due to subversion of the maternal immune tolerance and system immunity of the animals and the GRA15II effector contributes to the process of adverse pregnant consequences.

RevDate: 2019-08-09
CmpDate: 2019-08-09

Lane-Reticker SK, Manguso RT, WN Haining (2018)

Pooled in vivo screens for cancer immunotherapy target discovery.

Immunotherapy, 10(3):167-170.

RevDate: 2019-08-08

Kim JG, Garrett S, Wei Y, et al (2019)

CRISPR DNA elements controlling site-specific spacer integration and proper repeat length by a Type II CRISPR-Cas system.

Nucleic acids research pii:5545008 [Epub ahead of print].

CRISPR-Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3' hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.

RevDate: 2019-08-08

Ai L, Guo W, Chen W, et al (2019)

The gal80 Deletion by CRISPR-Cas9 in Engineered Saccharomyces cerevisiae Produces Artemisinic Acid Without Galactose Induction.

Current microbiology pii:10.1007/s00284-019-01752-2 [Epub ahead of print].

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has emerged as the dominating tool for genome engineering, while also changes the speed and efficiency of metabolic engineering in conventional and non-conventional yeasts. Among these CRISPR-Cas systems, CRISPR-Cas9 technology has usually been applied for removing unfavorable target genes. Here, we used CRISPR-Cas9 technology to delete the gal80 gene in uracil-deficient strain and had successfully remolded the engineered Saccharomyces cerevisiae that can produce artemisinic acid without galactose induction. An L9(34) orthogonal test was adopted to investigate the effects of different factors on artemisinic acid production. Fermentation medium III with sucrose as carbon sources, 1% inoculum level, and 84-h culture time were identified as the optimal fermentation conditions. Under this condition, the maximum artemisinic acid production by engineered S. cerevisiae 1211-2 was 740 mg/L in shake-flask cultivation level. This study provided an effective approach to reform metabolic pathway of artemisinic acid-producing strain. The engineered S. cerevisiae 1211-2 may be applied to artemisinic acid production by industrial fermentation in the future.

RevDate: 2019-08-08

Zhang F, Song G, Y Tian (2019)

Anti-CRISPRs: The natural inhibitors for CRISPR-Cas systems.

Animal models and experimental medicine, 2(2):69-75 pii:AME212069.

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR associated protein) systems serve as the adaptive immune system by which prokaryotes defend themselves against phages. It has also been developed into a series of powerful gene-editing tools. As the natural inhibitors of CRISPR-Cas systems, anti-CRISPRs (Acrs) can be used as the "off-switch" for CRISPR-Cas systems to limit the off-target effects caused by Cas9. Since the discovery of CRISPR-Cas systems, much research has focused on the identification, mechanisms and applications of Acrs. In light of the rapid development and scientific significance of this field, this review summarizes the history and research status of Acrs, and considers future applications.

RevDate: 2019-08-08

Martens KJA, van Beljouw SPB, van der Els S, et al (2019)

Visualisation of dCas9 target search in vivo using an open-microscopy framework.

Nature communications, 10(1):3552 pii:10.1038/s41467-019-11514-0.

CRISPR-Cas9 is widely used in genomic editing, but the kinetics of target search and its relation to the cellular concentration of Cas9 have remained elusive. Effective target search requires constant screening of the protospacer adjacent motif (PAM) and a 30 ms upper limit for screening was recently found. To further quantify the rapid switching between DNA-bound and freely-diffusing states of dCas9, we developed an open-microscopy framework, the miCube, and introduce Monte-Carlo diffusion distribution analysis (MC-DDA). Our analysis reveals that dCas9 is screening PAMs 40% of the time in Gram-positive Lactoccous lactis, averaging 17 ± 4 ms per binding event. Using heterogeneous dCas9 expression, we determine the number of cellular target-containing plasmids and derive the copy number dependent Cas9 cleavage. Furthermore, we show that dCas9 is not irreversibly bound to target sites but can still interfere with plasmid replication. Taken together, our quantitative data facilitates further optimization of the CRISPR-Cas toolbox.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Do PT, Nguyen CX, Bui HT, et al (2019)

Demonstration of highly efficient dual gRNA CRISPR/Cas9 editing of the homeologous GmFAD2-1A and GmFAD2-1B genes to yield a high oleic, low linoleic and α-linolenic acid phenotype in soybean.

BMC plant biology, 19(1):311 pii:10.1186/s12870-019-1906-8.

BACKGROUND: CRISPR/Cas9 gene editing is now revolutionizing the ability to effectively modify plant genomes in the absence of efficient homologous recombination mechanisms that exist in other organisms. However, soybean is allotetraploid and is commonly viewed as difficult and inefficient to transform. In this study, we demonstrate the utility of CRISPR/Cas9 gene editing in soybean at relatively high efficiency. This was shown by specifically targeting the Fatty Acid Desaturase 2 (GmFAD2) that converts the monounsaturated oleic acid (C18:1) to the polyunsaturated linoleic acid (C18:2), therefore, regulating the content of monounsaturated fats in soybean seeds.

RESULTS: We designed two gRNAs to guide Cas9 to simultaneously cleave two sites, spaced 1Kb apart, within the second exons of GmFAD2-1A and GmFAD2-1B. In order to test whether the Cas9 and gRNAs would perform properly in transgenic soybean plants, we first tested the CRISPR construct we developed by transient hairy root transformation using Agrobacterium rhizogenesis strain K599. Once confirmed, we performed stable soybean transformation and characterized ten, randomly selected T0 events. Genotyping of CRISPR/Cas9 T0 transgenic lines detected a variety of mutations including large and small DNA deletions, insertions and inversions in the GmFAD2 genes. We detected CRISPR- edited DNA in all the tested T0 plants and 77.8% of the events transmitted the GmFAD2 mutant alleles to T1 progenies. More importantly, null mutants for both GmFAD2 genes were obtained in 40% of the T0 plants we genotyped. The fatty acid profile analysis of T1 seeds derived from CRISPR-edited plants homozygous for both GmFAD2 genes showed dramatic increases in oleic acid content to over 80%, whereas linoleic acid decreased to 1.3-1.7%. In addition, transgene-free high oleic soybean homozygous genotypes were created as early as the T1 generation.

CONCLUSIONS: Overall, our data showed that dual gRNA CRISPR/Cas9 system offers a rapid and highly efficient method to simultaneously edit homeologous soybean genes, which can greatly facilitate breeding and gene discovery in this important crop plant.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Bertolini MS, Chiurillo MA, Lander N, et al (2019)

MICU1 and MICU2 Play an Essential Role in Mitochondrial Ca2+ Uptake, Growth, and Infectivity of the Human Pathogen Trypanosoma cruzi.

mBio, 10(3): pii:mBio.00348-19.

The mitochondrial Ca2+ uptake in trypanosomatids, which belong to the eukaryotic supergroup Excavata, shares biochemical characteristics with that of animals, which, together with fungi, belong to the supergroup Opisthokonta. However, the composition of the mitochondrial calcium uniporter (MCU) complex in trypanosomatids is quite peculiar, suggesting lineage-specific adaptations. In this work, we used Trypanosoma cruzi to study the role of orthologs for mitochondrial calcium uptake 1 (MICU1) and MICU2 in mitochondrial Ca2+ uptake. T. cruzi MICU1 (TcMICU1) and TcMICU2 have mitochondrial targeting signals, two canonical EF-hand calcium-binding domains, and localize to the mitochondria. Using the CRISPR/Cas9 system (i.e., clustered regularly interspaced short palindromic repeats with Cas9), we generated TcMICU1 and TcMICU2 knockout (-KO) cell lines. Ablation of either TcMICU1 or TcMICU2 showed a significantly reduced mitochondrial Ca2+ uptake in permeabilized epimastigotes without dissipation of the mitochondrial membrane potential or effects on the AMP/ATP ratio or citrate synthase activity. However, none of these proteins had a gatekeeper function at low cytosolic Ca2+ concentrations ([Ca2+]cyt), as occurs with their mammalian orthologs. TcMICU1-KO and TcMICU2-KO epimastigotes had a lower growth rate and impaired oxidative metabolism, while infective trypomastigotes have a reduced capacity to invade host cells and to replicate within them as amastigotes. The findings of this work, which is the first to study the role of MICU1 and MICU2 in organisms evolutionarily distant from animals, suggest that, although these components were probably present in the last eukaryotic common ancestor (LECA), they developed different roles during evolution of different eukaryotic supergroups. The work also provides new insights into the adaptations of trypanosomatids to their particular life styles.IMPORTANCETrypanosoma cruzi is the etiologic agent of Chagas disease and belongs to the early-branching eukaryotic supergroup Excavata. Its mitochondrial calcium uniporter (MCU) subunit shares similarity with the animal ortholog that was important to discover its encoding gene. In animal cells, the MICU1 and MICU2 proteins act as Ca2+ sensors and gatekeepers of the MCU, preventing Ca2+ uptake under resting conditions and favoring it at high cytosolic Ca2+ concentrations ([Ca2+]cyt). Using the CRISPR/Cas9 technique, we generated TcMICU1 and TcMICU2 knockout cell lines and showed that MICU1 and -2 do not act as gatekeepers at low [Ca2+]cyt but are essential for normal growth, host cell invasion, and intracellular replication, revealing lineage-specific adaptations.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Gulei D, Raduly L, Berindan-Neagoe I, et al (2019)

CRISPR-based RNA editing: diagnostic applications and therapeutic options.

Expert review of molecular diagnostics, 19(2):83-88.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Cornelissen LAM, Blanas A, van der Horst JC, et al (2019)

Disruption of sialic acid metabolism drives tumor growth by augmenting CD8+ T cell apoptosis.

International journal of cancer, 144(9):2290-2302.

Sialylated glycan structures are known for their immunomodulatory capacities and their contribution to tumor immune evasion. However, the role of aberrant sialylation in colorectal cancer and the consequences of complete tumor desialylation on anti-tumor immunity remain unstudied. Here, we report that CRISPR/Cas9-mediated knock out of the CMAS gene, encoding a key enzyme in the sialylation pathway, in the mouse colorectal cancer MC38 cell line completely abrogated cell surface expression of sialic acids (MC38-Sianull) and, unexpectedly, significantly increased in vivo tumor growth compared to the control MC38-MOCK cells. This enhanced tumor growth of MC38-Sianull cells could be attributed to decreased CD8+ T cell frequencies in the tumor microenvironment only, as immune cell frequencies in tumor-draining lymph nodes remained unaffected. In addition, MC38-Sianull cells were able to induce CD8+ T cell apoptosis in an antigen-independent manner. Moreover, low CMAS gene expression correlated with reduced recurrence-free survival in a human colorectal cancer cohort, supporting the clinical relevance of our work. Together, these results demonstrate for the first time a detrimental effect of complete tumor desialylation on colorectal cancer tumor growth, which greatly impacts the design of novel cancer therapeutics aimed at altering the tumor glycosylation profile.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Bruni GO, Zhong K, Lee SC, et al (2019)

CRISPR-Cas9 induces point mutation in the mucormycosis fungus Rhizopus delemar.

Fungal genetics and biology : FG & B, 124:1-7.

Rhizopus delemar causes devastating mucormycosis in immunodeficient individuals. Despite its medical importance, R. delemar remains understudied largely due to the lack of available genetic markers, the presence of multiple gene copies due to genome duplication, and mitotically unstable transformants resulting from conventional and limited genetic approaches. The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) system induces efficient homologous and non-homologous break points and generates individual and multiple mutant alleles without requiring selective marker genes in a wide variety of organisms including fungi. Here, we have successfully adapted this technology for inducing gene-specific single nucleotide (nt) deletions in two clinical strains of R. delemar: FGSC-9543 and CDC-8219. For comparative reasons, we first screened for spontaneous uracil auxotrophic mutants resistant to 5-fluoroorotic acid (5-FOA) and obtained one substitution (f1) mutationin the FGSC-9543 strain and one deletion (f2) mutation in the CDC-8219 strain. The f2 mutant was then successfully complemented with a pyrF-dpl200 marker gene. We then introduced a vector pmCas9:tRNA-gRNA that expresses both Cas9 endonuclease and pyrF-specific gRNA into FGSC-9543 and CDC-8219 strains and obtained 34 and 42 5-FOA resistant isolates, respectively. Candidate transformants were successively transferred eight times by propagating hyphal tips prior to genotype characterization. Sequencing of the amplified pyrF allele in all transformants tested revealed a single nucleotide (nt) deletion at the 4th nucleotide before the protospacer adjacent motif (PAM) sequence, which is consistent with CRISPR-Cas9 induced gene mutation through non-homologous end joining (NHEJ). Our study provides a new research tool for investigating molecular pathogenesis mechanisms of R. delemar while also highlighting the utilization of CRISPR-Cas9 technology for generating specific mutants of Mucorales fungi.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Przybilla MJ, Ou L, Tăbăran AF, et al (2019)

Comprehensive behavioral and biochemical outcomes of novel murine models of GM1-gangliosidosis and Morquio syndrome type B.

Molecular genetics and metabolism, 126(2):139-150.

Deficiencies in the lysosomal hydrolase β-galactosidase (β-gal) lead to two distinct diseases: the skeletal disease Morquio syndrome type B, and the neurodegenerative disease GM1-gangliosidosis. Utilizing CRISPR-Cas9 genome editing, the mouse β-gal encoding gene, Glb1, was targeted to generate both models of β-gal deficiency in a single experiment. For Morquio syndrome type B, the common human missense mutation W273L (position 274 in mice) was introduced into the Glb1 gene (Glb1W274L), while for GM1-gangliosidosis, a 20 bp mutation was generated to remove the catalytic nucleophile of β-gal (β-gal-/-). Glb1W274L mice showed a significant reduction in β-gal enzyme activity (8.4-13.3% of wildtype), but displayed no marked phenotype after one year. In contrast, β-gal-/- mice were devoid of β-gal enzyme activity (≤1% of wildtype), resulting in ganglioside accumulation and severe cellular vacuolation throughout the central nervous system (CNS). β-gal-/- mice also displayed severe neuromotor and neurocognitive dysfunction, and as the disease progressed, the mice became emaciated and succumbed to the disease by 10 months of age. Overall, in addition to generating a novel murine model that phenotypically resembles GM1-gangliosidosis, the first model of β-galactosidase deficiency with residual enzyme activity has been developed.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Liu X, Wang M, Qin Y, et al (2018)

Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9.

BMC biotechnology, 18(1):66.

BACKGROUND: Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired.

RESULTS: We generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17-0.45%) that was used to knock in an approximately 5-kb long DNA fragment.

CONCLUSION: We developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Tang JX, Chen D, Deng SL, et al (2018)

CRISPR/Cas9-mediated genome editing induces gene knockdown by altering the pre-mRNA splicing in mice.

BMC biotechnology, 18(1):61.

BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9 (CRISPR/Cas9) has been wildly used to generate gene knockout models through inducing indels causing frame-shift. However, there are few studies concerning the post-transcript effects caused by CRISPR-mediated genome editing.

RESULTS: In the present study, we showed that gene knockdown model also could be generated using CRISPR-mediated gene editing by disrupting the boundary of exon and intron in mice (C57BL/6 J). CRISPR induced indel at the boundary of exon and intron (5' splice site) caused alternative splicing and produced multiple different mRNAs, most of these mRNAs introduced premature termination codon causing down expression of the gene.

CONCLUSIONS: These results showed that alternative splicing mutants were able to generate through CRISPR-mediated genome editing by deleting the boundary of exon and intron causing disruption of 5' splice site. Although alternative splicing was an unexpected outcome, this finding could be developed as a technology to generate gene knockdown models or to investigate pre-mRNA splicing.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Chen D, Tang JX, Li B, et al (2018)

CRISPR/Cas9-mediated genome editing induces exon skipping by complete or stochastic altering splicing in the migratory locust.

BMC biotechnology, 18(1):60.

BACKGROUND: The CRISPR/Cas9 system has been widely used to generate gene knockout/knockin models by inducing frameshift mutants in cell lines and organisms. Several recent studies have reported that such mutants can lead to in-frame exon skipping in cell lines. However, there was little research about post-transcriptional effect of CRISPR-mediated gene editing in vivo.

RESULTS: We showed that frameshift indels also induced complete or stochastic exon skipping by deleting different regions to influence pre-mRNA splicing in vivo. In the migratory locust, the missing 55 bp at the boundary of intron 3 and exon 4 of an olfactory receptor gene, LmigOr35, resulted in complete exon 4 skipping, whereas the lacking 22 bp in exon 4 of LmigOr35 only resulted in stochastic exon 4 skipping. A single sgRNA induced small insertions or deletions at the boundary of intron and exon to disrupt the 3' splicing site causing completely exon skipping, or alternatively induce small insertions or deletions in the exon to stochastic alter splicing causing the stochastic exon skipping.

CONCLUSIONS: These results indicated that complete or stochastic exon skipping could result from the CRISPR-mediated genome editing by deleting different regions of the gene. Although exon skipping caused by CRISPR-mediated editing was an unexpected outcome, this finding could be developed as a technology to investigate pre-mRNA splicing or to cure several human diseases caused by splicing mutations.

RevDate: 2019-08-08
CmpDate: 2019-08-08

de Buhr H, RJ Lebbink (2018)

Harnessing CRISPR to combat human viral infections.

Current opinion in immunology, 54:123-129.

CRISPR/Cas9 is a technology that allows for targeted and precise genome editing in eukaryotic cells. The technique has changed the landscape of molecular biology and may be applied to repair genetic disorders in future therapies. Besides targeting the human genome, it can be used to cleave and edit viral DNA present in infected cells, and as such provides a promising new strategy for anti-viral therapy. Here, we discuss recent studies on the use of anti-viral CRISPRs to target pathogenic human viruses, with a focus on in vivo studies, challenges, and potential for future clinical applications.

RevDate: 2019-08-08
CmpDate: 2019-08-08

Allison SJ (2018)

Genetic engineering: Trans-epigenetic modulation of target genes in acute kidney injury.

Nature reviews. Nephrology, 14(2):72.

RevDate: 2019-08-07

Young CS, Pyle AD, MJ Spencer (2019)

CRISPR for Neuromuscular Disorders: Gene Editing and Beyond.

Physiology (Bethesda, Md.), 34(5):341-353.

This is a review describing advances in CRISPR/Cas-mediated therapies for neuromuscular disorders (NMDs). We explore both CRISPR-mediated editing and dead Cas approaches as potential therapeutic strategies for multiple NMDs. Last, therapeutic considerations, including delivery and off-target effects, are also discussed.

RevDate: 2019-08-07

Kim Y, Lee SJ, Yoon HJ, et al (2019)

Anti-CRISPR AcrIIC3 discriminates between Cas9 orthologs via targeting the variable surface of the HNH nuclease domain.

The FEBS journal [Epub ahead of print].

CRISPR-Cas systems constitute the adaptive immunity of bacteria and archaea, degrading nucleic acids of invading phages and plasmids. In response, phages employ anti-CRISPR (Acr) proteins as a counter-defense mechanism to neutralize the host immunity. AcrIIC3 directly inhibits target DNA cleavage of type II-C Cas9 of Neisseria meningitidis. Here we show that AcrIIC3 interacts with the HNH nuclease domain of N. meningitidis Cas9 to inhibit its nuclease activity in an allosteric manner. The crystal structure of the AcrIIC3-HNH complex reveals that AcrIIC3 binds opposite the active site on the HNH nuclease domain. AcrIIC3 employs a unique interface for HNH, allowing it to discriminate between Cas9 orthologs, which contrasts with the broad spectrum of Cas9 inhibition by AcrIIC1. Interface residues of HNH provide key electrostatic and hydrophobic interactions that determine the host specificity of AcrIIC3. Mutations that replace HNH interfaces of N. meningitidis Cas9 with those of Geobacillus stearothermophilus Cas9 or Campylobacter jejuni Cas9 significantly attenuate AcrIIC3 binding, illustrating that the divergent interaction surface confers the host specificity of AcrIIC3. Our study demonstrates that the variable sequences of binding interface can define the target specificity of Acr proteins, suggesting potential applications in Cas9 control for gene editing. This article is protected by copyright. All rights reserved.

RevDate: 2019-08-07
CmpDate: 2019-08-07

Chen G, J Chu (2019)

Characterization of Two Polyketide Synthases Involved in Sorbicillinoid Biosynthesis by Acremonium chrysogenum Using the CRISPR/Cas9 System.

Applied biochemistry and biotechnology, 188(4):1134-1144.

Acremonium chrysogenum is an important fungal strain used for cephalosporin C production. Many efforts have been made to develop versatile genome-editing tools to better understand the mechanism of A. chrysogenum. Here, we developed a feasible and efficient CRISPR/Cas9 system. Two genes responsible for the synthesis of yellow pigments (sorbicillinoids) were chosen as targets, and plasmids expressing both the Cas9 protein and single-guide RNAs were constructed. After introducing the plasmids into the protoplasts of A. chrysogenum, 83 to 93% albino mutants harboring the expected genomic alteration, on average, were obtained. We have generated two mutant strains that respectively disrupt sorA and sorB by flexible CRISPR/Cas9 system. We further confirmed that the sorbicillinoid biosynthetic gene cluster is regulated by an autoinduction mechanism. This work will lay a solid foundation for gene function research and regulation in the sorbicillinoid biosynthetic pathway.

RevDate: 2019-08-07
CmpDate: 2019-08-07

Zhou W, Cui H, Ying L, et al (2018)

Enhanced Cytosolic Delivery and Release of CRISPR/Cas9 by Black Phosphorus Nanosheets for Genome Editing.

Angewandte Chemie (International ed. in English), 57(32):10268-10272.

A biodegradable two-dimensional (2D) delivery platform based on loading black phosphorus nanosheets (BPs) with Cas9 ribonucleoprotein engineered with three nuclear localization signals (NLSs) at C terminus (Cas9N3) is successfully established. The Cas9N3-BPs enter cells effectively via membrane penetration and endocytosis pathways, followed by a BPs biodegradation-associated endosomal escape and cytosolic releases of the loaded Cas9N3 complexes. The Cas9N3-BPs thus provide efficient genome editing and gene silencing in vitro and in vivo at a relatively low dose as compared with other nanoparticle-based delivery platforms. This biodegradable 2D delivery platform offers a versatile cytosolic delivery approach for CRISPR/Cas9 ribonucleoprotein and other bioactive macromolecules for biomedical applications.

RevDate: 2019-08-07
CmpDate: 2019-08-07

Huang M, Inukai T, Miyake K, et al (2018)

Clofarabine exerts antileukemic activity against cytarabine-resistant B-cell precursor acute lymphoblastic leukemia with low deoxycytidine kinase expression.

Cancer medicine, 7(4):1297-1316.

Cytosine arabinoside (Ara-C) is one of the key drugs for the treatment of acute myeloid leukemia. It is also used for consolidation therapy of acute lymphoblastic leukemia (ALL). Ara-C is a deoxyadenosine analog and is phosphorylated to form cytosine arabinoside triphosphate (Ara-CTP) as an active form. In the first step of the metabolic pathway, Ara-C is phosphorylated to Ara-CMP by deoxycytidine kinase (DCK). However, the current cumulative evidence in the association of the Ara-C sensitivity in ALL appears inconclusive. We analyzed various cell lines for the possible involvement of DCK in the sensitivities of B-cell precursor ALL (BCP-ALL) to Ara-C. Higher DCK expression was associated with higher Ara-C sensitivity. DCK knockout by genome editing with a CRISPR-Cas9 system in an Ara-C-sensitive-ALL cell line induced marked resistance to Ara-C, but not to vincristine and daunorubicin, indicating the involvement of DCK expression in the Ara-C sensitivity of BCP-ALL. DCK gene silencing due to the hypermethylation of a CpG island and reduced DCK activity due to a nonsynonymous variant allele were not associated with Ara-C sensitivity. Clofarabine is a second-generation deoxyadenosine analog rationally synthesized to improve stability and reduce toxicity. The IC50 of clofarabine in 79 BCP-ALL cell lines was approximately 20 times lower than that of Ara-C. In contrast to Ara-C, although the knockout of DCK induced marked resistance to clofarabine, sensitivity to clofarabine was only marginally associated with DCK gene expression level, suggesting a possible efficacy of clofarabine for BCP-ALL that shows relative Ara-C resistance due to low DCK expression.

RevDate: 2019-08-02
CmpDate: 2019-08-02

McAllister KN, Bouillaut L, Kahn JN, et al (2017)

Using CRISPR-Cas9-mediated genome editing to generate C. difficile mutants defective in selenoproteins synthesis.

Scientific reports, 7(1):14672.

Clostridium difficile is a significant concern as a nosocomial pathogen, and genetic tools are important when analyzing the physiology of such organisms so that the underlying physiology/pathogenesis of the organisms can be studied. Here, we used TargeTron to investigate the role of selenoproteins in C. difficile Stickland metabolism and found that a TargeTron insertion into selD, encoding the selenophosphate synthetase that is essential for the specific incorporation of selenium into selenoproteins, results in a significant growth defect and a global loss of selenium incorporation. However, because of potential polar effects of the TargeTron insertion, we developed a CRISPR-Cas9 mutagenesis system for C. difficile. This system rapidly and efficiently introduces site-specific mutations into the C. difficile genome (20-50% mutation frequency). The selD CRISPR deletion mutant had a growth defect in protein-rich medium and mimicked the phenotype of a generated TargeTron selD mutation. Our findings suggest that Stickland metabolism could be a target for future antibiotic therapies and that the CRISPR-Cas9 system can introduce rapid and efficient modifications into the C. difficile genome.

RevDate: 2019-08-07
CmpDate: 2019-08-07

Casper J, Zweig AS, Villarreal C, et al (2018)

The UCSC Genome Browser database: 2018 update.

Nucleic acids research, 46(D1):D762-D769.

The UCSC Genome Browser (https://genome.ucsc.edu) provides a web interface for exploring annotated genome assemblies. The assemblies and annotation tracks are updated on an ongoing basis-12 assemblies and more than 28 tracks were added in the past year. Two recent additions are a display of CRISPR/Cas9 guide sequences and an interactive navigator for gene interactions. Other upgrades from the past year include a command-line version of the Variant Annotation Integrator, support for Human Genome Variation Society variant nomenclature input and output, and a revised highlighting tool that now supports multiple simultaneous regions and colors.

RevDate: 2019-08-06

Hajizadeh Dastjerdi A, Newman A, G Burgio (2019)

The Expanding Class 2 CRISPR Toolbox: Diversity, Applicability, and Targeting Drawbacks.

BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy pii:10.1007/s40259-019-00369-y [Epub ahead of print].

The class 2 clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, one of the prokaryotic adaptive immune systems, has sparked a lot of attention for its use as a gene editing tool. Currently, type II, V, and VI effector modules of this class have been characterized and extensively tested for nucleic acid editing, imaging, and disease diagnostics. Due to the unique composition of their nuclease catalytic center, the effector modules substantially vary in their function and possible biotechnology applications. In this review, we discuss the structural and functional diversity in class 2 CRISPR effectors, and debate their suitability for nucleic acid targeting and their shortcomings as gene editing tools.

RevDate: 2019-08-05

Wolter F, H Puchta (2019)

In planta gene targeting can be enhanced by the use of CRISPR/Cas12a.

The Plant journal : for cell and molecular biology [Epub ahead of print].

The controlled change of plant genomes by homologous recombination (HR) is still difficult to achieve. We developed the in planta gene targeting (ipGT) technology which depends on the simultaneous activation of the target locus by a DSB and the excision of the target vector. Whereas the use of SpCas9 resulted in low ipGT frequencies in Arabidopsis, we were recently able to improve the efficiency by using egg cell specific expression of the potent but less broadly applicable SaCas9 nuclease. We now tested whether we can improve ipGT further, by either performing it in cells with enhanced intrachromosomal HR efficiencies or by the use of Cas12a, a different kind of CRISPR/Cas nuclease with an alternative cutting mechanism. We could show before that plants possess three kinds of DNA ATPase complexes, which all lead to instabilities of homologous genomic repeats if lost by mutation. As these proteins act in independent pathways, we tested ipGT in double mutants in which intrachromosomal HR is enhanced 20 to 80 fold. However, we were not able to obtain higher ipGT frequencies, indicating that mechanisms for GT and chromosomal repeat-induced HR differ. However, using LbCas12a, the GT frequencies were higher than with SaCas9, despite a lower NHEJ induction efficiency, demonstrating the particular suitability of Cas12a to induce HR. As SaCas9 has substantial restrictions due to its longer GC rich PAM sequence, the use of LbCas12a with its AT-rich PAM broadens the range of ipGT drastically, particularly when targeting in CG-deserts like promoters and introns. This article is protected by copyright. All rights reserved.

RevDate: 2019-07-23

Lalonde S, Codina-Fauteux VA, de Bellefon SM, et al (2019)

Integrative analysis of vascular endothelial cell genomic features identifies AIDA as a coronary artery disease candidate gene.

Genome biology, 20(1):133 pii:10.1186/s13059-019-1749-5.

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD) and blood pressure (BP) or hypertension. Many of these loci are not linked to traditional risk factors, nor do they include obvious candidate genes, complicating their functional characterization. We hypothesize that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis, such as roles in forming a selective barrier, inflammation, hemostasis, and vascular tone, and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. To test this hypothesis, we generate an integrated map of gene expression, open chromatin region, and 3D interactions in resting and TNFα-treated human endothelial cells.

RESULTS: We show that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We identify physical loops by Hi-C and link open chromatin peaks that include CAD or BP SNPs with the promoters of genes expressed in endothelial cells. This analysis highlights 991 combinations of open chromatin regions and gene promoters that map to 38 CAD and 92 BP GWAS loci. We validate one CAD locus, by engineering a deletion of the TNFα-sensitive regulatory element using CRISPR/Cas9 and measure the effect on the expression of the novel CAD candidate gene AIDA.

CONCLUSIONS: Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD and hypertension.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Urnov FD (2019)

Hijack of CRISPR defences by selfish genes holds clinical promise.

Nature, 571(7764):180-181.

RevDate: 2019-07-23

Teng F, Guo L, Cui T, et al (2019)

CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity.

Genome biology, 20(1):132 pii:10.1186/s13059-019-1742-z.

CRISPR-based nucleic acid detection methods are reported to facilitate rapid and sensitive DNA detection. However, precise DNA detection at the single-base resolution and its wide applications including high-fidelity SNP genotyping remain to be explored. Here we develop a Cas12b-mediated DNA detection (CDetection) strategy, which shows higher sensitivity on examined targets compared with the previously reported Cas12a-based detection platform. Moreover, we show that CDetection can distinguish differences at the single-base level upon combining the optimized tuned guide RNA (tgRNA). Therefore, our findings highlight the high sensitivity and accuracy of CDetection, which provides an efficient and highly practical platform for DNA detection.

RevDate: 2019-07-08

Nishiki I, Yoshida T, A Fujiwara (2019)

Complete genome sequence and characterization of virulence genes in Lancefield group C Streptococcus dysgalactiae isolated from farmed amberjack (Seriola dumerili).

Microbiology and immunology, 63(7):243-250.

Lancefield group C Streptococcus dysgalactiae causes infections in farmed fish. Here, the genome of S. dysgalactiae strain kdys0611, isolated from farmed amberjack (Seriola dumerili) was sequenced. The complete genome sequence of kdys0611 consists of a single chromosome and five plasmids. The chromosome is 2,142,780 bp long and has a GC content of 40%. It possesses 2061 coding sequences and 67 tRNA and 6 rRNA operons. One clustered regularly interspaced short palindromic repeat, 125 insertion sequences, and four predicted prophage elements were identified. Phylogenetic analysis based on 126 core genes suggested that the kdys0611 strain is more closely related to S. dysgalactiae subsp. dysgalactiae than to S. dysgalactiae subsp. equisimilis. The genome of kdys0611 harbors 87 genes with sequence similarity to putative virulence-associated genes identified in other bacteria, of which 57 exhibit amino acid identity (>52%) to genes of the S. dysgalactiae subsp. equisimilis GGS124 human clinical isolate. Four putative virulence genes, emm5 (FGCSD_0256), spg_2 (FGCSD_1961), skc (FGCSD_1012), and cna (FGCSD_0159), in kdys0611 did not show significant homology with any deposited S. dysgalactiae genes. The chromosomal sequence of kdys0611 has been deposited in GenBank under Accession No. AP018726. This is the first report of the complete genome sequence of S. dysgalactiae isolated from fish.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Matsuo K, G Atsumi (2019)

CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins.

Planta, 250(2):463-473.

MAIN CONCLUSION: RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology. The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.

RevDate: 2019-04-19

Beneke T, E Gluenz (2019)

LeishGEdit: A Method for Rapid Gene Knockout and Tagging Using CRISPR-Cas9.

Methods in molecular biology (Clifton, N.J.), 1971:189-210.

Postgenomic analyses of Leishmania biology benefit from rapid and precise methods for gene manipulation. Traditional methods of gene knockout or tagging by homologous recombination have limitations: they tend to be slow and require successive transfection and selection rounds to knock out multiple alleles of a gene. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems overcome these limitations. We describe here in detail a simple, rapid, and scalable method for CRISPR-Cas9-mediated gene knockout and tagging in Leishmania. This method details how to use simple PCR to generate (1) templates for single guide RNA (sgRNA) transcription in cells expressing Cas9 and T7 RNA polymerase and (2) drug-selectable editing cassettes, using a modular set of plasmids as templates. pT plasmids allow for amplification of drug resistance genes for knockouts and pPLOT plasmids provide a choice of different tags to generate N- or C-terminally tagged proteins. We describe how to use an online platform (LeishGEdit.net) for automated primer design and how to perform PCRs and transfections in small batches or on 96-well plates for large-scale knockout or tagging screens. This method allows generation of knockout mutants or tagged cell lines within 1 week.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Kjos M (2019)

Transcriptional Knockdown in Pneumococci Using CRISPR Interference.

Methods in molecular biology (Clifton, N.J.), 1968:89-98.

Sequence-specific knockdown of gene expression using CRISPR interference (CRISPRi) has recently been developed for Streptococcus pneumoniae. By coexpression of a catalytically inactive Cas9-protein (dCas9) and a single guide RNA (sgRNA), CRISPRi can be used to knock down transcription of any gene of interest. Gene specificity is mediated by a 20 bp sequence on the sgRNA, and new genes can be targeted by replacing this 20 bp sequence. Here, a protocol is provided for design of sgRNAs and construction of CRIPSRi strains in S. pneumoniae, based on the vectors published by Liu et al. (Mol Syst Biol 13:931, 2017).

RevDate: 2019-08-05

Ledford H (2019)

Bull 'super dads' are being engineered to produce sperm from another father.

Nature, 567(7748):292-293.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Dong Y, Peng T, Wu W, et al (2019)

Efficient introduction of an isogenic homozygous mutation to induced pluripotent stem cells from a hereditary hearing loss family using CRISPR/Cas9 and single-stranded donor oligonucleotides.

The Journal of international medical research, 47(4):1717-1730.

BACKGROUND: Heterozygous purinergic receptor p2x gene (P2RX2) c.178G>T (p.V60L) mutations can lead to progressive hearing loss (HL) and increased susceptibility to noise. However, the underlying mechanisms remain unclear. A combination of human induced pluripotent stem cell (hiPSC) technology with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas)9-mediated gene editing may provide a promising tool to study gene function and treat hereditary deafness in humans.

METHODS: hiPSC technology and CRISPR/Cas9-mediated gene editing were used to generate heterozygous and homozygous P2RX2 c.178G>T (p.V60L) cell models.

RESULTS: We generated non-integrative hiPSCs from urine samples derived from three members of a large Chinese family carrying heterozygous P2RX2 c.178G>T mutations (designated P2RX2+/-) as a model to study P2RX2-mediated hereditary HL. Furthermore, we used CRISPR/Cas9 and single-stranded donor oligonucleotides to genetically establish homozygous P2RX2 c.178G>T hiPSCs (designated P2RX2-/-) from heterozygous patient-specific hiPSCs as a control to further study the pathological gene function.

CONCLUSIONS: Heterozygous and homozygous P2RX2-mutated hiPSC lines are good models to investigate the pathological mechanisms of P2RX2 mutations in HL pathogenesis. Our findings confirmed our hypothesis that it is feasible and convenient to introduce precise point mutations into genomic loci of interest to generate gene-mutated hiPSC models.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Karakus U, Thamamongood T, Ciminski K, et al (2019)

MHC class II proteins mediate cross-species entry of bat influenza viruses.

Nature, 567(7746):109-112.

Zoonotic influenza A viruses of avian origin can cause severe disease in individuals, or even global pandemics, and thus pose a threat to human populations. Waterfowl and shorebirds are believed to be the reservoir for all influenza A viruses, but this has recently been challenged by the identification of novel influenza A viruses in bats1,2. The major bat influenza A virus envelope glycoprotein, haemagglutinin, does not bind the canonical influenza A virus receptor, sialic acid or any other glycan1,3,4, despite its high sequence and structural homology with conventional haemagglutinins. This functionally uncharacterized plasticity of the bat influenza A virus haemagglutinin means the tropism and zoonotic potential of these viruses has not been fully determined. Here we show, using transcriptomic profiling of susceptible versus non-susceptible cells in combination with genome-wide CRISPR-Cas9 screening, that the major histocompatibility complex class II (MHC-II) human leukocyte antigen DR isotype (HLA-DR) is an essential entry determinant for bat influenza A viruses. Genetic ablation of the HLA-DR α-chain rendered cells resistant to infection by bat influenza A virus, whereas ectopic expression of the HLA-DR complex in non-susceptible cells conferred susceptibility. Expression of MHC-II from different bat species, pigs, mice or chickens also conferred susceptibility to infection. Notably, the infection of mice with bat influenza A virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant. Collectively, our data identify MHC-II as a crucial entry mediator for bat influenza A viruses in multiple species, which permits a broad vertebrate tropism.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Li Z, Zhou L, Jiang T, et al (2019)

Proteasomal deubiquitinase UCH37 inhibits degradation of β-catenin and promotes cell proliferation and motility.

Acta biochimica et biophysica Sinica, 51(3):277-284.

The ubiquitin-proteasome system degrades most cellular proteins in eukaryotes. UCH37, also known as UCH-L5, is a deubiquitinase binding to Rpn13, a receptor for ubiquitinated substrates in the 26 S proteasome. But, it remains unclear how UCH37 influences the proteasomal degradation of the ubiquitinated substrates. Because deletion of UCH37 is embryonically lethal in mice, this study aims to investigate the role of UCH37 in proteasomal degradation by constructing the UCH37-deficient cell lines using CRISPR/Cas9 technology. Our results demonstrated that deletion of UCH37 decreased the levels of proteasomal Rpn13, implying that UCH37 might facilitate incorporation of Rpn13 into the proteasome. Meanwhile, deletion of UCH37 decreased the levels of β-catenin and the early endosomal protein Rab8. β-Catenin interacts with TCF/LEF to control transcription, and is involved in development, tissue homeostasis and tumorigenesis. We further found that deletion of UCH37 increased the levels of the ubiquitinated β-catenin and accelerated the hydrogen peroxide-stimulated degradation of β-catenin. Deletion of UCH37 also down-regulated the transcription of c-Myc, a downstream effector of β-catenin, and inhibited cell proliferation and motility. These results raise the possibility that UCH37 maintains the homeostasis of proteasomal degradation reciprocally by assisting the recruitment of the ubiquitin receptor Rpn13 into the proteasome and by reversing ubiquitination of certain critical substrates of the 26 S proteasome.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Bi HL, Xu J, He L, et al (2019)

CRISPR/Cas9-mediated ebony knockout results in puparium melanism in Spodoptera litura.

Insect science, 26(6):1011-1019.

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.

RevDate: 2019-04-30

Li Y, Huang C, Zha L, et al (2019)

Generation of NERCe003-A-3, a p53 compound heterozygous mutation human embryonic stem cell line, by CRISPR/Cas9 editing.

Stem cell research, 34:101371.

p53 is a tumor suppressor gene involved mainly in the regulation of the G1/S cell cycle phase, DNA repair, and senescence. Although p53 is frequently altered in human cancer, the consequences of its depletion in human embryonic stem cells (hESCs) are unknown. We generated NERCe003-A-3, a p53 knockout hESC line, from the normal NERCe003-A hESC line by using CRISPR/Cas9 editing. This cell line maintained a normal 46, XY karyotype. Further analysis suggested that the cells expressed pluripotency-related markers and had the capacity to differentiate in vitro into derivatives of all three germ layers.

RevDate: 2019-04-30

Schuster S, Saravanakumar S, Schöls L, et al (2019)

Generation of a homozygous CRISPR/Cas9-mediated knockout human iPSC line for the STUB1 locus.

Stem cell research, 34:101378.

STUB1/CHIP is a central component of cellular protein homeostasis and interacts with key proteins involved in the pathogenesis of many neurodegenerative diseases. Missense and truncating mutations in STUB1 lead to SCAR16. For ideal in vitro disease modelling with isogenic controls, we generated a CHIP knockout cell line from a healthy control with no CHIP functionality, but remaining genomic integrity and verified pluripotency.

RevDate: 2019-04-30

Xue Y, Liao B, Xie Y, et al (2019)

Establishment of an ectodermal dysplasia related gene EDA Knockout human embryonic stem cell line (WAe001-A-22) by CRISPR-Cas9 technology.

Stem cell research, 34:101379.

EDA is a gene located at Xq13.1. It encodes different isoforms of tumor necrosis factor (TNF) superfamily member ectodysplasin A. Ectodysplasin A is a transmembrane protein which can be cleaved to form a secreted form and interact with EDA receptor to mediate the development of ectoderm. Mutations of the EDA gene are related to ectodermal dysplasia and tooth agenesis. Here, we report the establishment of the EDA gene knockout human embryonic stem (hES) cell line by CRISPR-Cas9 technology. This cell line provides good materials for further studies of the roles ectodysplasin A plays in ectoderm differentiation and tooth development.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Xu J, Chen RM, Chen SQ, et al (2019)

Identification of a germline-expression promoter for genome editing in Bombyx mori.

Insect science, 26(6):991-999.

Identification of stage- and tissue-specific cis-regulatory elements will enable more precise genomic editing. In previous studies of the silkworm Bombyx mori, we identified and characterized several tissue- and sex-specific cis-regulatory elements using transgenic technology, including a female- and fat body-specific promoter, vitellogenin, testis-specific promoters, Radial spoke head 1 (BmR1) and beta-tubulin 4 (Bmβ4). Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter, nanos (Bmnos). We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb, nos-1kb and nos-2kb) and in vivo (nos-2kb). In BmN cultured cells, all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP), although nos-2kb had the highest fluorescence activity. In transgenic silkworms, nos-2kb drove EGFP expression at the early embryonic stage, and fluorescence was concentrated in the gonads at later embryonic stages. In addition, this cis-regulatory element was not sex differentiated. The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body. The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter. These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Liu T, Yang WQ, Xie YG, et al (2019)

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus.

Insect science, 26(6):1045-1054.

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in G1 pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used in Ae. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.

RevDate: 2019-05-29

Damodharan S, Corem S, Gupta SK, et al (2018)

Tuning of SlARF10A dosage by sly-miR160a is critical for auxin-mediated compound leaf and flower development.

The Plant journal : for cell and molecular biology, 96(4):855-868.

miR160 adjusts auxin-mediated development by post-transcriptional regulation of the auxin response factors ARF10/16/17. In tomato, knockdown of miR160 (sly-miR160) suggested that it is required for auxin-driven leaf blade outgrowth, but whether additional developmental events are adjusted by sly-miR160 is not clear. Here, the SlMIR160 genes and the genes of its SlARFs targets were edited by CRISPR/Cas9 resulting in the isolation of loss-of-function mutants. In addition, hypomorphic mutants that accumulate variable reduced levels of sly-miR160a were isolated. We found that the loss-of-function mutants in SlMIR160a (CR-slmir160a-6/7) produced only four wiry leaves, whereas the hypomorphic mutants developed leaves and flowers with graded developmental abnormalities. Phenotypic severity correlated with the upregulation of SlARF10A. Consistent with that, double mutants in SlMIR160a and SlARF10A restored leaf and flower development indicating that over-accumulation of SlARF10A underlay the developmental abnormalities exhibited in the CR-slmir160a mutants. Phenotype severity also correlated with the upregulation of the SHOOT MERISTEMLESS homolog Tomato Knotted 2, which in turn activated the transcription of the cytokinin biosynthesis genes SlIPT2 and SlIPT4. However, no change in Tomato Knotted 2 was detected in the absence of SlARF10A, suggesting that it is upregulated due to auxin signaling suppression by SlARF10A. Knockout of sly-miR160a-targeted SlARFs showed that whereas SlARF10A is indispensable for leaf blade outgrowth and floral organ patterning, the functions of SlARF16A and SlARF17 are redundant. Taken together our results suggest that sly-miR160a promotes blade outgrowth as well as leaf and leaflet initiation and floral organ development through the quantitative regulation of its major target SlARF10A.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Wang YH, Chen XE, Yang Y, et al (2019)

The Masc gene product controls masculinization in the black cutworm, Agrotis ipsilon.

Insect science, 26(6):1037-1044.

Sex determination has been studied in the model lepidopteran species Bombyx mori, but it remains poorly understood in lepidopteran pests. In the present study, we identified and characterized the Masculinizer (Masc) gene in a Noctuidae pest species, Agrotis ipsilon. Sequence analysis revealed that AiMasc encodes a protein of 658 amino acids that has two CCCH-type zinc finger domains and two conserved cysteine residues (Cys-277 and Cys-280). We assessed the masculinizing activity of AiMasc in BmN cells and found that AiMasc induced expression of the male-specific doublesex isoform. Disruption of Masc via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in A. ipsilon caused abnormalities in abdominal segments and external genitalia, resulting in male-specific sterility. These results suggest that Masc participates in the process of sex determination in A. ipsilon. Successful identification of sex-determination gene in a pest species may enable the development of novel genetic approaches for pest control.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Wang XG, Ma SY, Chang JS, et al (2019)

Programmable activation of Bombyx gene expression using CRISPR/dCas9 fusion systems.

Insect science, 26(6):983-990.

The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have made it possible to reprogram target gene expression without cloning complementary DNA or disturbing genomic sequence in mammalian cells and several multicellular organisms. We previously showed that CRISPR-associated protein 9 (Cas9) and CRISPR from Prevotella and Francisella 1 (Cpf1) could induce target mutations, deletions, inversions, and duplications both singly and multiplex in silkworm, Bombyx mori. However, it remains unknown whether the CRISPR activation (CRISPRa) system can be used in B. mori. In this study, we investigated the CRISPRa system, in which a nuclease dead Streptococcus pyogenes Cas9 (SpCas9) is fused to two transcription activation domains, including VP64 (a tetramer of the herpes simplex VP16 transcriptional activator domain), and VPR (a tripartite activator, composed of VP64, p65, and Rta). The results showed that both dCas9-VP64 and dCas9-VPR systems could be used in B. mori cells, of which the latter showed significantly higher activity. The dCas9-VPR system showed considerable activity on all five tested target genes, and further analysis revealed that the up-regulation of genes was negatively correlated to their basal expression level. We also observed that this system could be used to upregulate a range of target genes. Taken together, our findings demonstrate that CRISPRa can be a powerful tool to study gene functions in B. mori and perhaps other non-drosophila insects.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Jia L, Ma SY, Zhang T, et al (2019)

Enhanced Bombyx genome editing via Cas9 ribonucleoprotein injection.

Insect science, 26(6):1059-1062.

RevDate: 2019-08-05
CmpDate: 2019-08-05

Bollen Y, Post J, Koo BK, et al (2018)

How to create state-of-the-art genetic model systems: strategies for optimal CRISPR-mediated genome editing.

Nucleic acids research, 46(13):6435-6454.

Model systems with defined genetic modifications are powerful tools for basic research and translational disease modelling. Fortunately, generating state-of-the-art genetic model systems is becoming more accessible to non-geneticists due to advances in genome editing technologies. As a consequence, solely relying on (transient) overexpression of (mutant) effector proteins is no longer recommended since scientific standards increasingly demand genetic modification of endogenous loci. In this review, we provide up-to-date guidelines with respect to homology-directed repair (HDR)-mediated editing of mammalian model systems, aimed at assisting researchers in designing an efficient genome editing strategy.


RJR Experience and Expertise


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.


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.


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.


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.


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.


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.


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.


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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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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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

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