I established a cell line stably expressing gRNA and Cas-9 using lentivirus. Do the sequence complementary to the gRNA sequence also get edited by Cas-9. Assuming the sequence complementary to the gRNA is undamaged, Cas-9 again has the opportunity to edit the previously edited site repeatedly. Under such scenario, is clonal screening worth a try?
The PAM sequence determines where Cas9 edits and causes the double strand break. If the sequence is edited successfully, it should theoretically no longer be edited since it no longer has the recognition site. During the repair, the other strand should be edited as well. If there are other alleles (i.e. diploid cells) then the other copy may not be edited since Cas9 never reached that site. That would be biallelic editing, though you would have to sequence to confirm strand specific editing.
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