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

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Moreb, EA; Lynch, MD

Published Date

  • August 19, 2021

Published In

Volume / Issue

  • 12 / 1

Start / End Page

  • 5034 -

PubMed ID

  • 34413309

Pubmed Central ID

  • PMC8377084

Electronic International Standard Serial Number (EISSN)

  • 2041-1723

International Standard Serial Number (ISSN)

  • 2041-1723

Digital Object Identifier (DOI)

  • 10.1038/s41467-021-25339-3

Language

  • eng