HDR ssDNA troubleshooting

I'm trying to correct a mutation through HDR using an antisense assymetric ssODN, with 36 nt on the PAM distal site and 91 bp on the PAM proximal site (as recommended by the Richardson 2017 paper).

I've introduced 1 silent mutation on the PAM but could only change from NGG to NAG, and 3 on the gRNA sequence (one on the PAM proximal site and 2 to generate a restriction site for RFLP analysis). If you add the point mutation for gene correction, that's 5 mutations in total on the template relative to the genomic target site.

When I tested the HDR knock-in in HEKs using a gRNA that cuts only 1 bp away from the mutation, I see no HDR at all after RFLP assay.

Is it possible that 5 mutations is too much? Is there a limit number to how much mutations we can introduce in the HDR template?

I'm going to test other HDR templates with different homology arm lengths, but I don't know wether I should reduce the number of silent mutations.

Thanks a lot for any thoughts you might share!


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Sand Werewolfover 4 years ago

5 mutations is a lot, we usually use 3 mutations total in the gRNA sequence (as close as possible to PAM site), and it works well.

RFLP may not be sensitive enough to pick up the modification. If you're rate is ~1%, that would be a very faint band to look for.

Also, try symmetric donors, sense and antisense if possible w 60bp arms, they work well for us. We usually see targeting rates ranging from 1-10% by NGS.

Good luck!

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