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

Péter István Kulcsár (), András Tálas, Eszter Tóth, Antal Nyeste, Zoltán Ligeti, Zsombor Welker and Ervin Welker ()
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Péter István Kulcsár: Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences
András Tálas: Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences
Eszter Tóth: Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences
Antal Nyeste: Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences
Zoltán Ligeti: Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences
Zsombor Welker: Biospiral-2006 Ltd
Ervin Welker: Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences

Nature Communications, 2020, vol. 11, issue 1, 1-14

Abstract: 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.

Date: 2020
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DOI: 10.1038/s41467-020-15021-5

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