New Evidence for the Effect of Human Chromatin on Cas9-Mediated Editing

The CRISPR/Cas9 system is a popular genome engineering tool that has the potential to be developed into a safe gene therapy, where errors in a patient’s genome can be precisely corrected with no off-target effects. By simply modifying the targeting sequence of the guide RNA, one can direct Cas9 to virtually any gene to generate mutations, deletions, or small insertions. However, bioengineers have yet to fully understand how the Cas9/RNA complex, a bacterial macromolecule, interacts with the complexities of a human genome that is packaged with nuclear proteins in a structure known as chromatin. So far, previous studies have only provided direct evidence that DNA methylation does not interfere with Cas9-mediated genome editing. However, genome-wide binding studies suggest Cas9 access to target DNA may be reduced by chromatin. The impact of the multiprotein complexes that make up the bulk of chromatin remains unclear. Using an inducible, transgenic cell line, we have directly measured chromatin inhibition of Cas9 editing efficiency at a reporter transgene that is stably integrated into a chromosome. Upon addition of doxycycline, the transgenic cells express a fusion protein that induces the accumulation of histone methylation, Polycomb complexes, and nucleosome compaction at a luciferase transgene. This switchable chromatin system allows us to compare the editing efficiency of a specific luciferase-targeted Cas9/gRNA complex in the presence and absence of compacted chromatin. We observed a decrease in editing efficiency at specific sites when chromatin is induced at the Cas9/gRNA target, luciferase. These results provide the first direct evidence that closed chromatin interferes with Cas9 function at a single site in the human genome.