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Blackjack mutations improve the on-target activities of increased fidelity variants of SpCas9 with 5′G-extended sgRNAs

2020; Nature Portfolio; Volume: 11; Issue: 1 Linguagem: Inglês

10.1038/s41467-020-15021-5

2041-1723

Péter István Kulcsár, András Tálas, Eszter Tóth, Antal Nyeste, Zoltán Ligeti, Zsombor Welker, Ervin Welker,

RNA Interference and Gene Delivery

Abstract 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.