I am trying to find all gRNAs in my target sequence of interest.

1.Am I right in thinking that provided that there is no annotation error I should get at least one hit with 0 mismatch for each generated gRNA, which correspond to my target sequence ?

2.Also, since my target sequence is very long I find it hard to discriminate between gRNAs - which criteria should I look for ? gRNA efficiency ? But as > 1 off targets are listed for each gRNAs, how should this value be used ? Should I be using the Top?OfftargetTotalScore in the Summary file instead ?

Thanks !

Maria, 

1. Yes. I suggest set max.mismatch = 0 and chromToSearch = "the target chromosome" to test this quickly

2.  Suggest use top?OfftargetTotalScore and efficacy score in the Summary file, and the detailed information in the offtarget output file such as whether the offtargets are in the exon of a gene and the number of offtargets.

Also if you are using spCas9, suggest set scoring.method = "CFDscore"

Best regards,

Julie

Thanks, really appreciate the help !