CRISPR with partially single-stranded PCR donor - maximum length?

Model Organism: Worms Scientific Discussion
1

Hi everyone,

Has anyone tried to increase the length of the inserts, using the partially single-stranded PCR donor as described in Dokshin et al., 2018? I’ve tried this method for fluorescent proteins and it works perfectly, up to 25% editing efficiencies in F1 rollers. However, I need a larger insert this time (~1100 bp) and I was wondering if anyone has tried larger inserts using this method?

Thanks in advance!

2

Absolutely

We didn’t have as high a rate of success but did manage to use it for knocking in several 3kb fragments and even a 6.5kb piece. So 1100bp should be easy. :wink:

Good luck!

Steve V