Minimize off-targets and improve Cripr/cas specificty

[Danioivanio]

Hi,

according to the last publication of Fu et al(Joung group) in Nature Biotech, they say that off-targets can be reduced efficiently by using 17 or 18 nt gRNA targetsites. So far so good. The publication of Seung Woo Cho et. al in Genome Research says that by using synthetic gRNA(IVT), chimeric gRNA and GGx20nt targetsites can minimize off target effects.

These two facts are contradicting each other in my opinion, maybe someone from the Joung group can give his opinion? I am looking forward to the answer.

Best
Ivan

So far the Crispr Cas in zebrafish is working great, also has a great germline transmission rate. KNOCK INs are possible by coinjecting plasmids(NHEJ repair). Great job so far in the community.

[pdh...@gmail.com]

hey Ivan, you will get more answers if you post in the Genome Engineering CRISPR forum.

but the short answer is - it seems that synthetic, in vitro transcribed sgRNA as opposed to U6-driven DNA encoding for sgRNA can improve specificity. this fits well with our understanding that the enzymatic concentration of Cas9/sgRNA is important for specificity (lower is more specific). and of course, a U6-driven plasmid will have much higher expression than transfected sgRNA