How to investigate off-target

[lh12565]

Hi, I have a normal and knock out (CRISPR) data from WGS. I want to investigate off-target effects. I run blast on sgRNA and get 60-100% identity sequences and location, a total of ~25000. I don't know how to use CRISPResso2 for evaluation and if the following command is correct:

CRISPRessoWGS -b hun2.sort.bam \
-f ko.txt \
-r Homo_sapiens_assembly38.fasta \
--gene_annotations gencode38.gz \
--name ko

ko.txt like:

chr11   16872937        16872953        LA-KO1   GATTTATACTCTTCTAA    NA     NA
chr16   79226054        79226043        LA-KO2   TGATTTATAATC    NA     NA
......
#a total of 25000

If it is correct, it may cause many results and graphs. Is there a simpler output and statistics to evaluate off-target effects? Thanks!

[kclem]

Yeah, that looks fine to me.

First, check that your region file is correct (e.g. the second line shows that the start coordinate is after the end coordinate).

The number of regions shouldn't be a problem. If you just care about quantification, you can run with the --suppress_plots or --suppress_report parameter which will run much faster and not produce plots.

Let me know if it works for you!

[lh12565]

Yeah, that looks fine to me.

First, check that your region file is correct (e.g. the second line shows that the start coordinate is after the end coordinate).

The number of regions shouldn't be a problem. If you just care about quantification, you can run with the --suppress_plots or --suppress_report parameter which will run much faster and not produce plots.

Let me know if it works for you!

Hi, @kclem , thank you for your quick reply. I have one more question. I don't want to output the "ANALYZED_REGIONS (folder)". It takes too long and doesn't make sense to me. How do I set parameters to avoid it? Thanks!

[kclem]

The ANALYZED_REGIONS folder is created as part of the processing. Would it work to just delete the folder after your run?