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

Author

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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)

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.

Suggested Citation

  • Zsolt Bodai & Alena L. Bishop & Valentino M. Gantz & Alexis C. Komor, 2022. "Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies," Nature Communications, Nature, vol. 13(1), pages 1-15, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-29989-9
    DOI: 10.1038/s41467-022-29989-9
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    1. Lukas Möller & Eric J. Aird & Markus S. Schröder & Lena Kobel & Lucas Kissling & Lilly van de Venn & Jacob E. Corn, 2022. "Recursive Editing improves homology-directed repair through retargeting of undesired outcomes," Nature Communications, Nature, vol. 13(1), pages 1-10, December.

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