Inhibition of CRISPR-Cas12a DNA targeting by nucleosomes and chromatin

Abstract

Genome engineering nucleases must access chromatinized DNA. Here, we investigate how AsCas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates target accessibility to Cas12a and determines the extent to which both steps of binding—PAM recognition and R-loop formation—are inhibited by the nucleosome. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing target-proximal dCas9 enhances DNA cleavage rates over 10-fold. Unexpectedly, Cas12a readily cleaves internucleosomal linker DNA within chromatin-like, phase-separated nucleosome arrays. DNA targeting is reduced only ~5-fold due to neighboring nucleosomes and chromatin compaction. This work explains the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted genomic regions in cells. We conclude that nucleosome unwrapping regulates accessibility to CRISPR-Cas nucleases and propose that increasing nucleosome breathing dynamics will improve DNA targeting in eukaryotic cells.

Genome-wide functional screens enable the prediction of high activity CRISPR-Cas9 and -Cas12a guides in Yarrowia lipolytica

“…The inclusion of nucleosome occupancy data improved Cas9 prediction accuracy, increasing the Pearson and Spearman r-values to 0.50 and 0.43, respectively. This effect is in agreement with observations of nucleosome inhibition of Cas9/12a targeting in vitro and in vivo [35][36][37][38] . A similar nucleosome occupancy effect on DeepGuide's ability to predict Cas12a CS values, however, was not observed here.…”

Demonstration of targeted crossovers in hybrid maize using CRISPR technology

“…Another explanation could be that the gRNA1 target site is not accessible to the guided nuclease at the early stages of plant regeneration. It is known that the activity of the guided nuclease is inhibited in the compact chromatin regions 22 – 24 . The chromatin modifications including histone and DNA methylation that modulate chromatin compactness are developmentally regulated 25 .…”

Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

“…Anecdotal reports from our own laboratory indicate that marker-free CRISPR editing at yeast telomeres is less efficient than elsewhere in the genome, likely because Cas9 cleavage occurs much less efficiently in this inaccessible heterochromatin context. While the studies above primarily focused on Cas9 activity in chromatin, a recent report indicates that the activity of other CRISPR enzymes ( i.e., Cas12a) can also be inhibited by nucleosomes in vitro ( Strohkendl et al, 2021 ). Taken together, these findings indicate that chromatin environments in living cells represent an important determinant for target specificity, as well as how successfully an intended genome editing outcome will be achieved.…”