Using flexiguides with CRISPResso2 pooled

[MyriamShafie]

Hello,

We are using CRISPResso2 pooled to map reads to multiple amplicons and it's working very nicely as long as the guide matches the amplicon perfectly. However, we also have cases where a guide is an imperfect match to an amplicon. There is an option in the regular CRISPResso2 to specify flexible guide sequences: can this option be used with CRISPResso2 pooled? If it is already possible, I couldn't figure out how. If not, it would be a really useful feature.

Thanks a lot!

[kclem]

Hi @MyriamShafie

Yes, we've thought about this but it turns out it is a somewhat hard problem -- how many mismatches do you allow between the guide and the off-target, and what if there are multiple best matches.

The pooled experiments we run include on- and off-targets, and the off-targets are computationally predicted. In the computational prediction part, the guide sequence at the off-target is produced, and this information can be directly fed into CRISPRessoPooled.

Is your use case one in which the guide sequences at off-targets are not known? Or is it an issue of converting the output of some computational prediction to CRISPRessoPooled input?

[MyriamShafie]

Hello @kclem,

Thanks for your answer. I'm not sure I completely understand your post but I'll try my best to explain the situation. We are working on wheat, which has three subgenomes. So, when we use a guide to target all three subgenomes at once, it often happens that a guide perfectly matches one or two of the subgenomes and has a couple of mismatches to the third. So the guide sequence is perfectly known, the three amplicon sequences (representing each subgenome) are also known and no cuts can really be called "off-target": it's just that we have multiple targets that we can't hit perfectly all at once. Hence our interest in using CRISPRessoPooled with guides that don't perfectly match an amplicon.