SignificanceThe efficiency of genome editing with CRISPR-Cas9 can vary widely at different targets and in different cells. Some of this variability may be due to the inherent quality of different guide RNAs, but it may also depend on the cellular context of the genomic target DNA. In this report, we demonstrate that targets bound by nucleosomes are cut much less efficiently than targets from which nucleosomes are absent or have been depleted. This information can inform target selection, particularly in cases where cells are quiescent or nucleosome mobility is limited.

CRISPR‐Cas9 is a prokaryotic immune surveillance complex, which has been adapted for use in eukaryotes as a revolutionizing RNA‐guided genome editing system. Despite its widespread success, differences in organization and structure of the prokaryotic versus eukaryotic genomes may limit its full utility. The eukaryotic genome is highly compact and organized into chromatin with stretches of DNA wrapped around histone protein cores (nucleosome). Additionally, for proper gene regulation, chromatin is further divided into nucleosome bound (NB) and nucleosome depleted regions (NDR). To make genome editing more efficient, we need to understand how chromatin may impact in vivo target accessibility and editing by CRISPR‐Cas9. Nucleosomes have been demonstrated to limit Cas9 accessibility in vitro, and indirect evidence suggests this may also be true in cells. To address this question directly, we are using Saccharomyces cerevisiae as a model system, as it has well‐characterized nucleosome locations and well worked out chromatin regulation. In particular, we are studying the cell cycle‐regulated HO (homothallic switching endonuclease) promoter in its native state as well as in mutants that contain constitutive NDRs in place of native NB regions. We find that several in vivo target sites within strongly positioned nucleosomes (NBs) are cut less efficiently than nearby nucleosome‐depleted regions (NDRs). Furthermore, when nucleosomes are displaced from NB targets by genetic manipulations, Cas9 cleavage efficiency increases. This indicates that reduced Cas9 cutting efficiency at NB targets is due to reduced accessibility to DNA rather than problems with Cas9 cleavage activity or poor guide‐RNA design. Developing an understanding of how genomic factors limit Cas9 accessibility will empower us to perform more precise and regulated genome modifications and to potentially design Cas9 variants with better chromatin access. Findings from the current study will help in making future genome targeting by CRISPR‐Cas9 more effective.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.