Hi,

I am working with the CRISPRseek module. I used both CFD and zhang scoring methods. I had two questions on the summary output file. 1. Why is that the summary output file include sgRNA which has a NA in topN.offtargettotalscore? Does it mean this sgRNA has no off target found? 2. Upon the topN.offtargettotalscore, I was thinking how the N was chosen? Is it related to the libray size we tried to investigate. For example, if we have 1000 sgRNAs, we select top100.offtargettotalscore; if we have 10000 sgRNAs, we select top1000.offtargettotalscore.

Please do help.

Thank you, Nan

Dear Nan,

Please see my response below.

1. Why does that the summary output file includes sgRNA which has a NA in topN.offtargettotalscore? Does it mean that this sgRNA has no off target found?

Yes, it means this sgRNA has no offtarget with the searching criteria you set. For example if you set max.mismatch = 3, then only offtargets with number of mismatch <= 3 will be considered.

Please feel free to explore the parameters by typing ?offTargetAnalysis in a R console.

1. Regarding topN.offtargettotalscore, I was thinking how to set the N. Is it related to the libray size we tried to investigate. For example, if we have 1000 sgRNAs, we select top100.offtargettotalscore; if we have 10000 sgRNAs, we select top1000.offtargettotalscore.

Very good question! topN.OfftargetTotalScore is not related to the number of sgRNAs in your library. Instead, it only affects the topN.offtargettotalscore in the summary page. The larger N you set, the larger the score is. If you are only interested in the top offtarget effects, then you set topN.offtargettotalscore to a smaller number. If you would like to know the overall offtarget effects, then you set topN.offtargettotalscore to a higher number.

If offtargets are a big concern to you, you will need to look at the offtargets.xls output file for the detailed information of the offtargets. Here are a couple of things you would like to pay special attention to in the offtargets.xls. Are there a few offtargets that dominate the topN.OfftargetTotalscore? Are those offtargets in the exon region of a gene? Does the offtarget have perfect PAM?

Hope this is helpful. Thanks!

Best regards,

Julie