Increased fidelity mutants of the SpCas9 nuclease constitute the most promising approach
to mitigating its off-target effects. However, these variants are effective only in
a restricted target space, and many of them are reported to work less efficiently
when applied in clinically relevant, pre-assembled, ribonucleoprotein forms. The low
tolerance to 5′-extended, 21G-sgRNAs contributes, to a great extent, to their decreased
performance. Here, we report the generation of Blackjack SpCas9 variant that shows
increased fidelity yet remain effective with 21G-sgRNAs. Introducing Blackjack mutations
into previously reported increased fidelity variants make them effective with 21G-sgRNAs
and increases their fidelity. Two “Blackjack” nucleases, eSpCas9-plus
and SpCas9-HF1-plus are superior variants of eSpCas9 and
SpCas9-HF1, respectively, possessing matching on-target activity and fidelity but
retaining activity with 21G-sgRNAs. They facilitate the use of existing pooled sgRNA
libraries with higher specificity and show similar activities whether delivered as
plasmids or as pre-assembled ribonucleoproteins.
