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      In silico Method in CRISPR/Cas System: An Expedite and Powerful Booster

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          The CRISPR/Cas system has stood in the center of attention in the last few years as a revolutionary gene editing tool with a wide application to investigate gene functions. However, the labor-intensive workflow requires a sophisticated pre-experimental and post-experimental analysis, thus becoming one of the hindrances for the further popularization of practical applications. Recently, the increasing emergence and advancement of the in silico methods play a formidable role to support and boost experimental work. However, various tools based on distinctive design principles and frameworks harbor unique characteristics that are likely to confuse users about how to choose the most appropriate one for their purpose. In this review, we will present a comprehensive overview and comparisons on the in silico methods from the aspects of CRISPR/Cas system identification, guide RNA design, and post-experimental assistance. Furthermore, we establish the hypotheses in light of the new trends around the technical optimization and hope to provide significant clues for future tools development.

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          Most cited references 180

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          Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

          In comparative high-throughput sequencing assays, a fundamental task is the analysis of count data, such as read counts per gene in RNA-seq, for evidence of systematic changes across experimental conditions. Small replicate numbers, discreteness, large dynamic range and the presence of outliers require a suitable statistical approach. We present DESeq2, a method for differential analysis of count data, using shrinkage estimation for dispersions and fold changes to improve stability and interpretability of estimates. This enables a more quantitative analysis focused on the strength rather than the mere presence of differential expression. The DESeq2 package is available at Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0550-8) contains supplementary material, which is available to authorized users.
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            CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.

            Horizontal gene transfer (HGT) in bacteria and archaea occurs through phage transduction, transformation, or conjugation, and the latter is particularly important for the spread of antibiotic resistance. Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci confer sequence-directed immunity against phages. A clinical isolate of Staphylococcus epidermidis harbors a CRISPR spacer that matches the nickase gene present in nearly all staphylococcal conjugative plasmids. Here we show that CRISPR interference prevents conjugation and plasmid transformation in S. epidermidis. Insertion of a self-splicing intron into nickase blocks interference despite the reconstitution of the target sequence in the spliced mRNA, which indicates that the interference machinery targets DNA directly. We conclude that CRISPR loci counteract multiple routes of HGT and can limit the spread of antibiotic resistance in pathogenic bacteria.
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              Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library.

              Identification of genes influencing a phenotype of interest is frequently achieved through genetic screening by RNA interference (RNAi) or knockouts. However, RNAi may only achieve partial depletion of gene activity, and knockout-based screens are difficult in diploid mammalian cells. Here we took advantage of the efficiency and high throughput of genome editing based on type II, clustered, regularly interspaced, short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems to introduce genome-wide targeted mutations in mouse embryonic stem cells (ESCs). We designed 87,897 guide RNAs (gRNAs) targeting 19,150 mouse protein-coding genes and used a lentiviral vector to express these gRNAs in ESCs that constitutively express Cas9. Screening the resulting ESC mutant libraries for resistance to either Clostridium septicum alpha-toxin or 6-thioguanine identified 27 known and 4 previously unknown genes implicated in these phenotypes. Our results demonstrate the potential for efficient loss-of-function screening using the CRISPR-Cas9 system.

                Author and article information

                Front Oncol
                Front Oncol
                Front. Oncol.
                Frontiers in Oncology
                Frontiers Media S.A.
                02 October 2020
                : 10
                1Hwa Mei Hospital, University of Chinese Academy of Science , Ningbo, China
                2Zhejiang Key Laboratory of Pathophysiology, Department of Preventative Medicine, Medical School of Ningbo University , Ningbo, China
                3Ningbo Institute of Life and Health Industry, University of Chinese Academy of Sciences , Ningbo, China
                Author notes

                Edited by: Meng Zhou, Wenzhou Medical University, China

                Reviewed by: Feizhen Wu, Fudan University, China; Wei Li, Children's National Hospital, United States

                *Correspondence: Ting Cai caiting@

                This article was submitted to Cancer Genetics, a section of the journal Frontiers in Oncology

                Copyright © 2020 Zhang, Zhao, Ahmed, Yi, Hu, Cai and Liao.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                Page count
                Figures: 4, Tables: 6, Equations: 0, References: 181, Pages: 20, Words: 13572
                Funded by: National Natural Science Foundation of China 10.13039/501100001809
                Funded by: Natural Science Foundation of Ningbo 10.13039/100007834


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