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Feb. 1, 2017 What is the CRISPR/Cas9 target site prediction program recommended for P. pacificus? Is it possible to obtain the GFF3 genome file for the chopchop site?
Ray H.
Dec. 17, 2016 Thanks Hanh. Was the ssDNA template on the same strand as the two sgRNAs? Did the deletion extend in both directions beyond each of the sgRNA target sites?
Ray
12.12.16 protocols for nucleosome isolation and agar/liquid culture
Michael
2016.12.09 @Ray: We undertook several attempts to knock out a larger region using two sgRNAs. In two cases sgRNAs were located a few kb and app. 500 bp apart, respectively, and we did not get any large deletions. Recently, we tried it with sgRNAs located app. 200 bp apart, and we also added a 100 bp long ssDNA repair template having 50 bp overlap upstream of the first cutting site and 50 bp overlap downstream of the second cutting site. The idea that we had in mind was that the repair template would ‘stitch together’ two large fragments of DNA during HDR. Interestingly, we did not get the deletion which we aimed for (app. 200 bp) but we got a larger deletion in this locus instead (app. 500 bp).
Hanh
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