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Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity

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  • E. A. Moreb

    (Duke University)

  • M. D. Lynch

    (Duke University)

Abstract

CRISPR-Cas9 is a powerful DNA editing tool. A gRNA directs Cas9 to cleave any DNA sequence with a PAM. However, some gRNA sequences mediate cleavage at higher efficiencies than others. To understand this, numerous studies have screened large gRNA libraries and developed algorithms to predict gRNA sequence dependent activity. These algorithms do not predict other datasets as well as their training dataset and do not predict well between species. Here, to better understand these discrepancies, we retrospectively examine sequence features that impact gRNA activity in 44 published data sets. We find strong evidence that gRNA sequence dependent activity is largely influenced by the ability of the Cas9/gRNA complex to find the target site rather than activity at the target site and that this drives sequence dependent differences in gRNA activity between different species. This understanding will help guide future work to understand Cas9 activity as well as efforts to identify optimal gRNAs and improve Cas9 variants.

Suggested Citation

  • E. A. Moreb & M. D. Lynch, 2021. "Genome dependent Cas9/gRNA search time underlies sequence dependent gRNA activity," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-25339-3
    DOI: 10.1038/s41467-021-25339-3
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    Cited by:

    1. Dipankar Baisya & Adithya Ramesh & Cory Schwartz & Stefano Lonardi & Ian Wheeldon, 2022. "Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    2. Dalton T. Ham & Tyler S. Browne & Pooja N. Banglorewala & Tyler L. Wilson & Richard K. Michael & Gregory B. Gloor & David R. Edgell, 2023. "A generalizable Cas9/sgRNA prediction model using machine transfer learning with small high-quality datasets," Nature Communications, Nature, vol. 14(1), pages 1-16, December.
    3. Giulia I. Corsi & Kunli Qu & Ferhat Alkan & Xiaoguang Pan & Yonglun Luo & Jan Gorodkin, 2022. "CRISPR/Cas9 gRNA activity depends on free energy changes and on the target PAM context," Nature Communications, Nature, vol. 13(1), pages 1-14, December.

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