pegRNA design¶
recommendations from Liu recent protocol.
https://www.nature.com/articles/s41596-022-00724-4
epegRNA is always better than traditional pegRNA
PE has much less off-targets, cas9-dependent or cas9-independent.
for 3’ motif / RNA motif that protects exonuclease degradation, try tevopreQ1 motif first.
sequences of spacer, PBS, RTT should avoid using polyA tracks, then try mpknot, csy4, zika, etc.
use unmodified scaffold first
RTT, at least 7nt downstream flanking. for longer indel, this flanking sequence should be even longer, e.g., 20nt.
PBS, initially testing 10, 13, 15nt.