Cas9 Activity in Human Chromatin: Inhibition and Strategies for Increased Efficiency

The prokaryotic CRISPR/Cas9 system enables precise changes at genomic targets. Due to the ease of its customization, Cas9 promises to make high-throughput genetic modifications possible. However, as Cas9 is a bacterially derived system, it has not evolved to interact with the mammalian genome which is compartmentalized and packaged into a nucleo-protein complex known as chromatin. We investigated the mechanism by which dynamic human chromatin states impede efficient Cas9 editing and identified a potential strategy to improve Cas9 efficiency. Using a transgenic HEK293 cell line, we induced accumulation of a Polycomb-based repressor (Gal4-EED) at a luciferase reporter gene to achieve partially and fully silenced chromatin states. We used a transactivator (Gal4-p65) to stimulate a hyperactive state. At silenced luciferase, we confirmed accumulation of the silencing mark H3K27me3 along luciferase by ChIP-qPCR. We measured Cas9-mediated editing using SURVEYOR assay and Cas9 binding using ChIP-qPCR. Interestingly, of the nine gRNA target sites tested along luciferase, Cas9 binding and cutting were inhibited at just five of the sites; four of the target sites were edited at a similar efficiency in each chromatin state. We also observed that a hyperactive state driven by Gal4-p65 reduces Cas9 editing, suggesting high expression levels could be a barrier to efficient Cas9 targeting. To determine a strategy for improving CRISPR efficiency, we broadly inhibited silenced chromatin with either siRNA against Polycomb or the chromatin disrupting drug UNC1999. Increased luciferase expression, indicating an active open chromatin state, was accompanied by increased Cas9 editing efficiency. Finally, we have designed a new transgenic system (inducible Gal4-chromatin remodeler fusions) to determine whether a DNA-targeted chromatin-remodeling proteins can open chromatin at a single locus for more precise enhancement of CRISPR accessibility. Ongoing work includes mapping of nucleosomes at CRISPR target sites to gain a deeper understanding of Cas9-chromatin interaction in human cells. So far, our work represents a significant advance towards understanding the mechanism and extent of Cas9-inhibition by dynamic chromatin. Artificial chromatin remodeling offers a promising solution for efficient binding and editing at sites located in silenced chromatin.