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bioRxiv · 10.1101/2020.07.21.213827

Precise and programmable C:G to G:C base editing in genomic DNA

Chen, L.·Park, J. E.·Paa, P.·Rajakumar, P. D.·Chew, Y. T.·Manivannan, S. N.·Chew, W. L.
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Abstract

Many genetic diseases are caused by single-nucleotide polymorphisms (SNPs). Base editors can correct SNPs at single-nucleotide resolution, but until recently, only allowed for C:G to T:A and A:T to G:C transition edits, addressing four out of twelve possible DNA base substitutions. Here we developed a novel class of C:G to G:C Base Editors (CGBEs) to create single-base genomic transversions in human cells. Our CGBEs consist of a nickase CRISPR-Cas9 (nCas9) fused to a cytosine deaminase and base excision repair (BER) proteins. Characterization of >30 CGBE candidates and 27 guide RNAs (gRNAs) revealed that CGBEs predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean C:G to G:C edits at 15% and up to 37%). CGBEs target cytosine in WCW, ACC or GCT sequence contexts and within a precise two-nucleotide window of the target protospacer. We further targeted genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of CGBE in interrogating and correcting human genetic diseases.

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Chen, L., Park, J. E., Paa, P., Rajakumar, P. D., Chew, Y. T., Manivannan, S. N., Chew, W. L.. 2020-07-21. Precise and programmable C:G to G:C base editing in genomic DNA. https://doi.org/10.1101/2020.07.21.213827

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