CRISPRseek (version 1.12.0)

CRISPRseek-package: Design of target-specific guide RNAs (gRNAs) in CRISPR-Cas9, genome-editing systems

Description

Design of target-specific gRNAs for the CRISPR-Cas9 system by automatically finding potential gRNAs (paired/not paired), with/without restriction enzyme cut site(s) in a given sequence, searching for off targets with user defined maximum number of mismatches, calculating score of each off target based on mismatch positions in the off target and a penalty weight matrix, filtering off targets with user-defined criteria, and annotating off targets with flank sequences, whether located in exon or not. Summary report is also generated with gRNAs ranked by total topN off target score, annotated with restriction enzyme cut sites, gRNA efficacy and possible paired gRNAs. Detailed paired gRNAs information and restriction enzyme cut sites are stored in separate files in the output directory specified by the user. In total, four tab delimited files are generated in the output directory: OfftargetAnalysis.xls (off target details), Summary.xls (gRNA summary), REcutDetails.xls (restriction enzyme cut sites of each gRNA), and pairedgRNAs.xls (potential paired gRNAs).

Details

Package:

CRISPRseek

Type:

Package

Version:

1.0

Date:

2013-10-04

License:

GPL (>= 2)

Function offTargetAnalysis integrates all steps of off target analysis into one function call

References

Mali P, Aach J, Stranges PB, Esvelt KM, Moosburner M, Kosuri S, Yang L, Church GM.CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol. 2013. 31(9):833-8 Patrick D Hsu, David A Scott, Joshua A Weinstein, F Ann Ran, Silvana Konermann, Vineeta Agarwala, Yinqing Li, Eli J Fine, Xuebing Wu, Ophir Shalem, Thomas J Cradick, Luciano A Marraffini, Gang Bao & Feng Zhang. DNA targeting specificity of rNA-guided Cas9 nucleases. Nat Biotechnol. 2013. 31:827-834 Lihua Julie Zhu, Benjamin R. Holmes, Neil Aronin and Michael Brodsky. CRISPRseek: a Bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems. Plos One Sept 23rd 2014 Doench JG et al., Optimized sgRNA design to maximize activity and minimize off-target effe cts of CRISPR-Cas9. Nature Biotechnology Jan 18th 2016

See Also

offTargetAnalysis

Examples

    library(CRISPRseek)
    library("BSgenome.Hsapiens.UCSC.hg19")
    library(TxDb.Hsapiens.UCSC.hg19.knownGene)
    library(org.Hs.eg.db)
    outputDir <- getwd()
    inputFilePath <- system.file("extdata", "inputseq.fa", package = "CRISPRseek")
    REpatternFile <- system.file("extdata", "NEBenzymes.fa", package = "CRISPRseek")
######## Scenario 1. Target and off-target analysis for paired gRNAs with 
######## one of the pairs overlap RE sites
    results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly=TRUE,
        REpatternFile =REpatternFile,findPairedgRNAOnly=TRUE, 
        BSgenomeName=Hsapiens, txdb=TxDb.Hsapiens.UCSC.hg19.knownGene, 
        orgAnn = org.Hs.egSYMBOL,max.mismatch = 1, chromToSearch = "chrX",
        outputDir = outputDir,overwrite = TRUE)

######## Scenario 2. Target and off-target analysis for paired gRNAs with or 
######## without RE sites
    results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE,
        REpatternFile = REpatternFile,findPairedgRNAOnly = TRUE,
        BSgenomeName = Hsapiens, txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, 
        orgAnn = org.Hs.egSYMBOL,max.mismatch = 1, chromToSearch = "chrX",
        outputDir = outputDir, overwrite = TRUE)

######## Scenario 3. Target and off-target analysis for gRNAs overlap RE sites

    results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = TRUE,
        REpatternFile = REpatternFile,findPairedgRNAOnly = FALSE, 
        BSgenomeName = Hsapiens, txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, 
        orgAnn = org.Hs.egSYMBOL, max.mismatch = 1, chromToSearch = "chrX", 
        outputDir = outputDir, overwrite = TRUE)

######## Scenario 4. Off-target analysis for all potential gRNAs, this will 
########be the slowest among the aforementioned scenarios.

    results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = FALSE, 
        REpatternFile = REpatternFile,findPairedgRNAOnly = FALSE,
        BSgenomeName = Hsapiens, txdb = TxDb.Hsapiens.UCSC.hg19.knownGene, 
         orgAnn = org.Hs.egSYMBOL, max.mismatch = 1, chromToSearch = "chrX", 
        outputDir = outputDir,overwrite = TRUE)

######## Scenario 5. Target and off-target analysis for gRNAs input by user.
    gRNAFilePath <- system.file("extdata", "testHsap_GATA1_ex2_gRNA1.fa",
        package="CRISPRseek")
    results <- offTargetAnalysis(inputFilePath = gRNAFilePath, findgRNAs = FALSE, 
        findgRNAsWithREcutOnly = FALSE, REpatternFile = REpatternFile,
        findPairedgRNAOnly = FALSE, BSgenomeName = Hsapiens, 
        txdb = TxDb.Hsapiens.UCSC.hg19.knownGene,
         orgAnn = org.Hs.egSYMBOL, max.mismatch = 1, chromToSearch = "chrX", 
        outputDir = outputDir, overwrite = TRUE)

####### Scenario 6. Quick gRNA finding  without target and off-target analysis 
    results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = TRUE,
        REpatternFile = REpatternFile,findPairedgRNAOnly = TRUE,  
        chromToSearch = "", outputDir = outputDir, overwrite = TRUE)

####### Scenario 7. Quick gRNA finding  with gRNA efficacy analysis
    results <- offTargetAnalysis(inputFilePath, findgRNAsWithREcutOnly = TRUE,
        REpatternFile = REpatternFile,findPairedgRNAOnly = TRUE,
	BSgenomeName = Hsapiens, annotateExon = FALSE,
        max.mismatch = 0, outputDir = outputDir, overwrite = TRUE)