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

Moreb, E. A.; Lynch, M. D.

doi:10.1038/s41467-021-25339-3

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

Journal Article · 18 August 2021 · Nature Communications

DOI: https://doi.org/10.1038/s41467-021-25339-3 · OSTI ID:1814211

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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.

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Research Organization:: Duke Univ., Durham, NC (United States)

Sponsoring Organization:: US Department of the Navy, Office of Naval Research (ONR); USDOE; USDOE Office of Energy Efficiency and Renewable Energy (EERE)

Grant/Contract Number:: EE0007563

OSTI ID:: 1814211

Journal Information:: Nature Communications, Journal Name: Nature Communications Journal Issue: 1 Vol. 12; ISSN 2041-1723

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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

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