CRISPRseek (version 1.12.0)

offTargetAnalysisOfPeakRegions: Finding offtargets in given regions

Description

Finding offtargets in given regions, such as peaks from GUIDE-seq

Usage

offTargetAnalysisOfPeakRegions(gRNA, peaks,  format=c("fasta", "bed"), peaks.withHeader = FALSE, BSgenomeName, upstream = 50, downstream =50, PAM.size = 3, gRNA.size = 20, PAM = "NGG", PAM.pattern = "NNN$", max.mismatch = 6, outputDir, allowed.mismatch.PAM = 3, overwrite = TRUE, weights = c(0, 0, 0.014, 0, 0, 0.395,
0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615,
0.804, 0.685, 0.583) )

Arguments

gRNA

gRNA input file path or a DNAStringSet object that contains gRNA plus PAM sequences used for genome editing

peaks

peak input file path or a GenomicRanges object that contains genomic regions to be searched for potential offtargets

format

Format of the gRNA and peak input file. Currently, fasta and bed are supported for gRNA and peak input file respectively

peaks.withHeader

Indicate whether the peak input file contains header, default FALSE

PAM.size

PAM length, default 3

gRNA.size

The size of the gRNA, default 20

PAM

PAM sequence after the gRNA, default NGG

BSgenomeName

BSgenome object. Please refer to available.genomes in BSgenome package. For example, BSgenome.Hsapiens.UCSC.hg19 for hg19, BSgenome.Mmusculus.UCSC.mm10 for mm10, BSgenome.Celegans.UCSC.ce6 for ce6, BSgenome.Rnorvegicus.UCSC.rn5 for rn5, BSgenome.Drerio.UCSC.danRer7 for Zv9, and BSgenome.Dmelanogaster.UCSC.dm3 for dm3

max.mismatch

Maximum mismatch allowed in off target search, default 6

PAM.pattern

Regular expression of protospacer-adjacent motif (PAM), default N[A|G]G$

allowed.mismatch.PAM

Number of degenerative bases in the PAM sequence, default to 2 for N[A|G]G PAM

outputDir

the directory where the off target analysis and reports will be written to

upstream

upstream offset from the peak start, default 50

downstream

downstream offset from the peak end, default 50

weights

a numeric vector size of gRNA length, default c(0, 0, 0.014, 0, 0, 0.395, 0.317, 0, 0.389, 0.079, 0.445, 0.508, 0.613, 0.851, 0.732, 0.828, 0.615, 0.804, 0.685, 0.583) which is used in Hsu et al., 2013 cited in the reference section

overwrite

overwrite the existing files in the output directory or not, default FALSE

Value

Same as compare2Sequences with one more tab-delimited file offTargetsInPeakRegions.xls containing all input peaks with potential gRNA binding sites, mismatch number and positions, and predicted cleavage score.

Details

References

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 (2013) DNA targeting specificity of rNA-guided Cas9 nucleases. Nature Biotechnology 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

See Also

CRISPRseek

Examples

   library(CRISPRseek)
   library("BSgenome.Hsapiens.UCSC.hg19")
   peaks <- system.file("extdata", "T2plus100OffTargets.bed",
      package = "CRISPRseek")
   gRNAs <- system.file("extdata", "T2.fa",
      package = "CRISPRseek")
   outputDir = getwd() 
   offTargets <- offTargetAnalysisOfPeakRegions(gRNA = gRNAs, peaks = peaks,
      format=c("fasta", "bed"),
      peaks.withHeader = TRUE, BSgenomeName = Hsapiens,
      upstream = 50, downstream =50, PAM.size = 3, gRNA.size = 20,
      PAM = "NGG", PAM.pattern = "NNN$", max.mismatch = 8,
      outputDir = outputDir,
      allowed.mismatch.PAM = 3, overwrite = TRUE
   )