TIDE analysis for Cpf1

[Gus McFarlane]

Hi all,

I am wondering how people are running their TIDE analyses for Cpf1. The online tool that I use - https://tide.nki.nl - is optimised for Cas9. Is there alternate software available for TIDE analysis for Cpf1 cleavage?

Thanks,

Gus

[Alex Ling]

Couldn't you just hack it by designating the "Cas9 guide" such that overlaps the Cpf1 cut site?

[Mike]

Actually, you don't even have to be that clever. As long as you provide it with a 20nt guide sequence that appears in the control file (possibly needs to have the NGG guide in the right place too, I'm not sure), TIDE will run. The only thing that will be incorrect is the predicted location of the cut site, but if you just ignore this, the predicted indels should be correct. I found this out by accident one day when I entered the wrong guide. As I was using two guides in the same exon., the guide was still in my control sequence, and everything ran fine, but just the predicted cut site was wrong.

Give it a test first though, I could be wrong!

[Gus McFarlane]

Thanks Mike and Alex. I'll get my sequencing back on Monday and give it a go.

Since posting I have also this seen on the TIDE website:

"Can TIDE be used for other nucleases (e.g. TALEN, ZFN, other RNA guided nucleases with different PAM)?

You can use TIDE for other RNA guided nucleases by entering in the TIDE webtool the DNA sequence around the expected cut site. TIDE assumes that a dsDNA break is induced between nucleotides 17 and 18 in the input sequence. You can estimate which sequence this will be for the other nucleases. Note that if you don’t know the exact breakpoint, TIDE should work fine to estimate the efficiency, but the +1 estimation is not be reliable anymore."

[Joann Wu]

Hi,

TIDE can be used for any DNA nuclease, as long as you know where the DNA break occurs. Even then, that may not be necessary, as long as you adjust your decomposition window correctly. The program only uses the traces in the set decomposition window.

I have used TIDE for Cpf1, and it works beautifully.

Joann

[Mark]

Thanks Joann, I'm just wondering what length are your Cpf1 gRNAs and where do you see the cuts relative to the PAM? I'm curious about the distribution.

[Joann Wu]

Hi,

I've tested published DNMT1-1 and DNMT1-3 gRNAs (that had been assessed previously by Zhang, Joung, and Kim), so the guides I tested were 23nt.

Similar to previous findings, I found the cut to occur ~18-19 nt downstream of the PAM.  

Joann

[Mark]

Joann, thanks for the reply. Sounds like a good place to start.