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

Nature Communications
Péter István KulcsárErvin Welker

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.

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Citations

Sep 9, 2020·Nature Structural & Molecular Biology·Heng ZhangLeifu Chang
Aug 28, 2020·Frontiers in Oncology·Fathema UddinTriparna Sen

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Datasets Mentioned

BETA
PRJNA593843

Methods Mentioned

BETA
X-ray
GUIDE-seq
transgenic
sgRNA
Assay
PCR
transfection
FACS
flow cytometry
transfections

Software Mentioned

GUIDE
samtools
BBMap
BoxPlotR
guideseq
Sniper
Image Lab
NEBuilder
BioPython
seq

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