README.md

In jianhong/CPD: CRISPR/Cas9 gRNA designer

CPD

CRISPR/Cas9 gRNA designer

Introduction

CPD is a R/Bioconductor package for CRISPR/Cas9 gRNA design. The input is GenomicRanges object. Output will be multiple excel sheet of primers and off targets. This is a parse of CRISPRseek. By input a bed file, users can search gRNA in upstream or downstream of the coordinates.

Installation

library(devtools)
install_github("jianhong/CPD")

Usage

Install dependence packages

Now it is support mm10, mm9, hg19, hg38, danRer10. Here is the packages should be installed for each assembly.

| | genome | txdb | orgAnn | -------- | ------------------------------- | ------------------------------------ | ------------ | mm10 | BSgenome.Mmusculus.UCSC.mm10 | TxDb.Mmusculus.UCSC.mm10.knownGene | org.Mm.eg.db | mm9 | BSgenome.Mmusculus.UCSC.mm9 | TxDb.Mmusculus.UCSC.mm9.knownGene | org.Mm.eg.db | hg19 | BSgenome.Hsapiens.UCSC.hg19 | TxDb.Hsapiens.UCSC.hg19.knownGene | org.Hs.eg.db | hg38 | BSgenome.Hsapiens.UCSC.hg38 | TxDb.Hsapiens.UCSC.hg38.knownGene | org.Hs.eg.db | danRer10 | BSgenome.Drerio.UCSC.danRer10 | TxDb.Drerio.UCSC.danRer10.refGene | org.Dr.eg.db

For example: for mm10

library(BiocInstaller)
biocLite(c("BSgenome.Mmusculus.UCSC.mm10", "TxDb.Mmusculus.UCSC.mm10.knownGene", "org.Mm.eg.db"))

Default

By default, the gRNA search is setting as upstream = 2000, downstream = 2000, scoring.method = "CFDscore", annotatePaired = FALSE, max.mismatch=1.

scoring.method = "CFDscore" means not only the mismatch position but also the mismatch type is considered for scoring (see https://www.nature.com/articles/nbt.3437). annotatePaired = FALSE means do NOT search potential paired gRNAs. max.mismatch =1 means max mismatch of off target position is 1.

## load the library
library(CPD)
## set the bed file path
gr <- system.file('extdata', 'Lamp3.bed', package = 'CPD')
## search gRNA
## anchor: Anchor point of upstream and downstream. 
##         set to TSS: search gRNA for promoter of given coordinates.
##         set to TES: search gRNA for upstream and downstream of transcript end site.
##         set to ALL: search gRNA for all region including upstream, body and downstream.
##         set to onlyUpstream&Downstream: search gRNA for upstream and downstream of given region (without the given region).
gRNA(gr, anchor="onlyUpstream&Downstream", species="mm10", outputDir="test")

Finding gRNAs with given restriction enzyme cut site(s)

The restriction enzyme cut sites could be set by REpatternFile parameter. It is the file path of given enzymes in fasta file. The content of restriction enzyme cut site pattern file will be like this:

BamHI
GGATCC
EcoRI
GAATTC

If the file name of the pattern file path is path/to/enzyme.fa, set REpatternFile="path/to/enzyme.fa".

gRNA(gr, anchor="onlyUpstream&Downstream", species="mm10", outputDir="test", REpatternFile="path/to/enzyme.fa")

Quick gRNA finding without off-target analysis

Set max.mismatch = 0 to find gRNAs without off-target analysis.

gRNA(gr, anchor="onlyUpstream&Downstream", species="mm10", outputDir="test", max.mismatch=0)

More

Get more helps such as how to change PAM pattern, gRNA pattern, please refer the vignette and help files of https://bioconductor.org/packages/release/bioc/html/CRISPRseek.html.

Output

In output folder, you will get multiple files.

1. gRNAsCRISPRseek.bed : file ready to be used for UCSC genome browser.

2. inputs.fa : fasta file could be used for gRNA search by other tools.

3. Summary.xls: gRNA summary

4. REcutDetails.xls: restriction enzyme cut sites of each gRNA

5. OfftargetAnalysis.xls: off-target details

6. pairedgRNAs.xls (optional): potential paired gRNAs

7. on.target.summary.csv: on target gRNA summary

How to read Summary.xls

names : name of gRNA

forViewInUCSC : coordinates

extendedSequence : extended sequence of gRNA

gRNAefficacy : gRNA efficacy. The higher the better. see https://www.nature.com/articles/nbt.3026

gRNAsPlusPAM : sequence of gRNAs plus PAM

top5OfftargetTotalScore : total score of top 5 offtarget. The higher the possibility to get off targets.

top10OfftargetTotalScore : total score of top 10 offtarget

top1Hit.onTarget.MMdistance2PAM : mismatch distance to PAM of the top 1 hit on target

topOfftargetNMMdistance2PAM : mismatch distance to PAM of the top N hit off target

REname : restriction enzyme name

uniqREin200 : unique restriction enzyme names in upstream 100 and downstream 100 around the gRNA.

Merge top N gRNAs from multiple on.target.summary.csv file

topN <- topNgRNA(n=5)

jianhong/CPD documentation built on May 4, 2019, 8:40 a.m.