Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies

Zsolt Bodai, Alena L. Bishop, Valentino M. Gantz and Alexis C. Komor ()
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Zsolt Bodai: University of California San Diego
Alena L. Bishop: University of California San Diego
Valentino M. Gantz: University of California San Diego
Alexis C. Komor: University of California San Diego

Nature Communications, 2022, vol. 13, issue 1, 1-15

Abstract: Abstract Programmable double-strand DNA breaks (DSBs) can be harnessed for precision genome editing through manipulation of the homology-directed repair (HDR) pathway. However, end-joining repair pathways often outcompete HDR and introduce insertions and deletions of bases (indels) at the DSB site, decreasing precision outcomes. It has been shown that indel sequences for a given DSB site are reproducible and can even be predicted. Here, we report a general strategy (the “double tap” method) to improve HDR-mediated precision genome editing efficiencies that takes advantage of the reproducible nature of indel sequences. The method simply involves the use of multiple gRNAs: a primary gRNA that targets the wild-type genomic sequence, and one or more secondary gRNAs that target the most common indel sequence(s), which in effect provides a “second chance” at HDR-mediated editing. This proof-of-principle study presents the double tap method as a simple yet effective option for enhancing precision editing in mammalian cells.

Date: 2022
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DOI: 10.1038/s41467-022-29989-9

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