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Old 07-24-2014, 01:40 AM   #1
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Location: London

Join Date: May 2012
Posts: 6
Default Finding CRISPR gRNA off-target matches using NGS mappers

Hi all,

To keep it brief I have about 3 million, 23bp sequences (gRNAs) which are putative CRIPSR-Cas9 sites, all of which I need to screened for off-target sites against the hg19 genome. I'm looking for mappings with up to 4 mis-matches, but for ALL alignments to be reported.

I've been using an R package called CRISPRseek, but to scan each gRNA would take ages, so I thought I would look at using NGS mappers to do the job.

The Sanger use bwa with these params:

bwa aln -n 5 -o 0 -l 20 -k 4 -N -m 1000000000 ${GENOME} ${CRISPR_FQ_FILE}

I tried this using just one gRNA, and the number of hits I got back were about 150 fewer than the R package function (350 vs 500). I manually looked at some sequences bwa missed, and I see no reason why bwa would reject them.

The question is whether there is another aligner I could consider? STAR will not work, and I don't think bowtie will allow up to 4mm. Alternatively, if there is a tweak to the above params I would be happy to hear it.


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