offTargetAnalysisOfPeakRegions: Offtarget Analysis of GUIDE-seq peaks

Description

Finding offtargets around peaks from GUIDE-seq or around any given genomic regions

Usage

offTargetAnalysisOfPeakRegions(gRNA, peaks, format=c("fasta", "bed"), peaks.withHeader = FALSE, BSgenomeName, overlap.gRNA.positions = c(17,18), upstream = 50, downstream =50, PAM.size = 3, gRNA.size = 20, PAM = "NGG", PAM.pattern = "(NAG|NGG|NGA)$", max.mismatch = 6, outputDir, allowed.mismatch.PAM = 2, 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), orderOfftargetsBy = c("predicted_cleavage_score", "n.mismatch"), descending = c(TRUE, FALSE), keepTopOfftargetsOnly = TRUE )

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

overlap.gRNA.positions

The required overlap positions of gRNA and restriction enzyme cut site, default 17 and 18 for SpCas9.

max.mismatch

Maximum mismatch allowed in off target search, default 6

PAM.pattern

Regular expression of protospacer-adjacent motif (PAM), default (NAG|NGG|NGA)$ for off target search

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 to search for off targets, default 50

downstream

downstream offset from the peak end to search for off targets, default 50

overwrite

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

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) for SPcas9 system, which is used in Hsu et al., 2013 cited in the reference section. Please make sure that the number of elements in this vector is the same as the gRNA.size, e.g., pad 0s at the beginning of the vector.

orderOfftargetsBy

criteria to order the offtargets by. By default, order by predicted_cleavage_score (descending order) followed by n.mismatch (ascending order) User can change the order of these two criteria and change descending order accordingly

descending

In the descending or ascending order. Default to order by predicted cleavage score in descending order and number of mismatch in ascending order When altering orderOfftargetsBy order, please also modify descending accordingly

keepTopOfftargetsOnly

Output all offtargets or the top offtarget using the orderOfftargetsBy criteria, default to the top offtarget

Value

a tab-delimited file offTargetsInPeakRegions.tsv, containing all input peaks with potential gRNA binding sites, mismatch number and positions, alignment to the input gRNA and predicted cleavage score.

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 Lihua Julie Zhu (2015). Overview of guide RNA design tools for CRISPR-Cas9 genome editing technology. Frontiers in Biology August 2015, Volume 10, Issue 4, pp 289-296

Examples

Run this code

if (interactive()) {
    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 = 2,
        outputDir = outputDir,
        allowed.mismatch.PAM = 3, overwrite = TRUE
   )
}