Will CRISPResso work for my reads of varying lengths?

[sarakn97]

Hello, I will be using CRISPResso2 to analyze the CRISPR-cas9 editing efficiency of my samples. I am using two sgRNAs and in some, I expect the 100 bp region between the sgRNAs will be completely deleted while it will not in others. The ones with the large deletion will be around 150 bp and the others will be 300 bp. These are paired-end reads. Will running CRISPResso2 be able to recognize this large deletion along with the other reads?

[kclem]

Hi @sarakn97,

Yes - CRISPResso should be able to handle this. There is a parameter --default_min_aln_score which sets the minimum alignment score. If the score is lower than this, reads will be discarded. By default this is 60%, meaning that at least 200/300 bases must match. So I would change this threshold to something lower like 50 or 40 like --default_min_aln_score 40.

You may also want to quantify the rate of creation of the large deletion. In this case, I'd provide the long (300bp) and short (150bp) amplicon as reference amplicons so that you can quantify the rate of long deletion creation as well as the indels produced around the cut sites in the long and short products like this:

CRISPResso --amplicon_seq AATTCCT{long}GGTT,AATT{short}TT --amplicon_name long,short ...

Let me know if that works.

Kendell

[sarakn97]

Thank you for the suggestions! I will let you know the results when I receive my sequenced samples back!