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      sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites

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          Abstract

          Although the CRISPR/Cas9/sgRNA system efficiently cleaves intracellular DNA at desired target sites, major concerns remain on potential “off-target” cleavage that may occur throughout the whole genome. In order to improve CRISPR-Cas9 specificity for targeted genome editing and transcriptional control, we describe a bioinformatics tool “sgRNAcas9”, which is a software package developed for fast design of CRISPR sgRNA with minimized off-target effects. This package consists of programs to perform a search for CRISPR target sites (protospacers) with user-defined parameters, predict genome-wide Cas9 potential off-target cleavage sites (POT), classify the POT into three categories, batch-design oligonucleotides for constructing 20-nt (nucleotides) or truncated sgRNA expression vectors, extract desired length nucleotide sequences flanking the on- or off-target cleavage sites for designing PCR primer pairs to validate the mutations by T7E1 cleavage assay. Importantly, by identifying potential off-target sites in silico, the sgRNAcas9 allows the selection of more specific target sites and aids the identification of bona fide off-target sites, significantly facilitating the design of sgRNA for genome editing applications. sgRNAcas9 software package is publicly available at BiooTools website ( www.biootools.com) under the terms of the GNU General Public License.

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          CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.

          Luciano A. Marraffini, Erik Sontheimer (2008)
          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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            Efficient multiplex biallelic zebrafish genome editing using a CRISPR nuclease system.

            Li-En Jao, Susan Wente, Wenbiao Chen (2013)
            A simple and robust method for targeted mutagenesis in zebrafish has long been sought. Previous methods generate monoallelic mutations in the germ line of F0 animals, usually delaying homozygosity for the mutation to the F2 generation. Generation of robust biallelic mutations in the F0 would allow for phenotypic analysis directly in injected animals. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. Here we report an improved CRISPR/Cas system in zebrafish with custom guide RNAs and a zebrafish codon-optimized Cas9 protein that efficiently targeted a reporter transgene Tg(-5.1mnx1:egfp) and four endogenous loci (tyr, golden, mitfa, and ddx19). Mutagenesis rates reached 75-99%, indicating that most cells contained biallelic mutations. Recessive null-like phenotypes were observed in four of the five targeting cases, supporting high rates of biallelic gene disruption. We also observed efficient germ-line transmission of the Cas9-induced mutations. Finally, five genomic loci can be targeted simultaneously, resulting in multiple loss-of-function phenotypes in the same injected fish. This CRISPR/Cas9 system represents a highly effective and scalable gene knockout method in zebrafish and has the potential for applications in other model organisms.
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              CRISPR interference (CRISPRi) for sequence-specific control of gene expression.

              Matthew Larson, Luke A Gilbert, Xiaowo Wang … (2013)
              Sequence-specific control of gene expression on a genome-wide scale is an important approach for understanding gene functions and for engineering genetic regulatory systems. We have recently described an RNA-based method, CRISPR interference (CRISPRi), for targeted silencing of transcription in bacteria and human cells. The CRISPRi system is derived from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the coexpression of a catalytically inactive Cas9 protein and a customizable single guide RNA (sgRNA). The Cas9-sgRNA complex binds to DNA elements complementary to the sgRNA and causes a steric block that halts transcript elongation by RNA polymerase, resulting in the repression of the target gene. Here we provide a protocol for the design, construction and expression of customized sgRNAs for transcriptional repression of any gene of interest. We also provide details for testing the repression activity of CRISPRi using quantitative fluorescence assays and native elongating transcript sequencing. CRISPRi provides a simplified approach for rapid gene repression within 1-2 weeks. The method can also be adapted for high-throughput interrogation of genome-wide gene functions and genetic interactions, thus providing a complementary approach to RNA interference, which can be used in a wider variety of organisms.
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                Author and article information

                Contributors
                Arkady B. Khodursky: Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (iso-abbrev): PLoS ONE
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2014
                Publication date (Electronic): 23 June 2014
                Volume: 9
                Issue: 6
                Electronic Location Identifier: e100448
                Affiliations
                [1 ]Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
                [2 ]MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, Nanjing, China
                [3 ]Shanghai Institute of Planned Parenthood Research, Shanghai, China
                University of Minnesota, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: SSX BS XXH. Performed the experiments: BS. Analyzed the data: CBZ. Contributed reagents/materials/analysis tools: SSX. Contributed to the writing of the manuscript: SSX YLZ XXH.

                Article
                Publisher ID: PONE-D-14-16485
                DOI: 10.1371/journal.pone.0100448
                PMC ID: 4067335
                PubMed ID: 24956386
                SO-VID: 89179619-5419-4999-87cb-69081743a568
                Copyright statement: Copyright @ 2014
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                Date received : 12 April 2014
                Date accepted : 23 May 2014
                Page count
                Pages: 9
                Funding
                The Project was supported by the National Natural Science Foundation of China (Grant No. 31301226) and the Chinese Academy of Sciences (CAS) Knowledge Innovation Program (KSXC2-EW-R-07). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Subject: Research Article
                Subject: Biology and Life Sciences
                Subject: Biochemistry
                Subject: Nucleic Acids
                Subject: Biotechnology
                Subject: Genetic Engineering
                Subject: Genetically Modified Organisms
                Subject: Transgenic Engineering
                Subject: Computational Biology
                Subject: Genetics
                Subject: Mutation
                Subject: Gene Deletion
                Subject: Genomics
                Subject: Genome Analysis
                Subject: Genome Scans
                Subject: Spacer Regions
                Subject: Animal Genomics
                Subject: Functional Genomics
                Subject: DNA
                Subject: Gene Disruption
                Subject: Gene Expression
                Subject: Gene Function
                Subject: Genetic Interference
                Subject: Computer and Information Sciences
                Subject: Computer Applications
                Subject: Computer-Aided Design
                Subject: Computer Software
                Subject: Open Source Software
                Subject: Software Engineering
                Subject: Software Design
                Subject: Software Tools
                Subject: Source Code
                Custom metadata
                Data Availability The authors confirm that all data underlying the findings are fully available without restriction. sgRNAcas9 software package is publicly available at BiooTools (Biological online tools) website www.biootools.com under the terms of the GPL license.

                ScienceOpen disciplines: Uncategorized
                Data availability:
                ScienceOpen disciplines: Uncategorized

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