Inhibition of CRISPR-Cas12a DNA Targeting by Nucleosomes and Chromatin

Abstract

Genome engineering nucleases, including CRISPR-Cas12a, must access chromatinized DNA. Here, we investigate how Acidaminococcus sp. Cas12a cleaves DNA within human nucleosomes and phase-condensed nucleosome arrays. Using quantitative kinetics approaches, we show that dynamic nucleosome unwrapping regulates DNA target accessibility to Cas12a. Nucleosome unwrapping determines the extent to which both steps of Cas12a binding-PAM recognition and R-loop formation-are inhibited by the nucleosome. Nucleosomes inhibit Cas12a binding even beyond the canonical core particle. Relaxing DNA wrapping within the nucleosome by reducing DNA bendability, adding histone modifications, or introducing a target-proximal nuclease-inactive Cas9 enhances DNA cleavage rates over 10-fold. Surprisingly, Cas12a readily cleaves DNA linking nucleosomes within chromatin-like phase separated nucleosome arrays-with DNA targeting reduced only ~4-fold. This work provides a mechanism for the observation that on-target cleavage within nucleosomes occurs less often than off-target cleavage within nucleosome-depleted regions of cells. We conclude that nucleosome wrapping restricts accessibility to CRISPR-Cas nucleases and anticipate that increasing nucleosome breathing dynamics will improve DNA binding and cleavage 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.…”

Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing

“…However, relative to microbial cells, the more complex cellular environments of mammalian cells may make it harder to cleave DNA. 10,11 To that end, we developed MIDAS to concurrently enhance the interactions between Cas nucleases and the PAM duplex (EIP), and the interactions between the catalytic pocket and ssDNA substrate (EIS), aiming for facilitating both DNA recognition and hydrolysis (Figure 1B). Given that these two processes are both required and reciprocally affect each other, we anticipated that MIDAS would dramatically enhance gene-editing activity in mammalian cells via a synergistic effect (Figure 1B).…”

Gene editing with CRISPR-Cas12a guides possessing ribose-modified pseudoknot handles

“…Converting predicted 2′-OH polar contact positions back to RNA usually rescues gene editing 70,71 . Thus, it appears that intrinsic biochemical activity is not dependent on 2′-OH contacts in bulk reactions in vitro but these become critical within cells, perhaps due to low biochemical RNP concentrations, more complex chromosomal DNA targets, or other unexplored biochemical or cellular factors 107 . Experiments like precision biochemical, thermodynamic, and structural studies, as well as sophisticated single-molecule or gene editing assays in cells, may be necessary to fully understand this phenomenon and unlock "RNA-free" CRISPR-Cas editing for advanced therapeutic control.…”