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.

Fig. 1. DNA unwrapping regulates Cas12a cleavage of nucleosomal targets.

(A) The Widom 601 positioning sequence is divided into quartiles indicating the inner (II and III) and outer (I and IV) wrap and is flanked by DNA. “****”: four TA dinucleotide repeats that produce tight wrapping of the inner left quartile. Arrows: Cas12a targets, pointing in the direction of R-loop formation; for top arrows, R-loop forms with the Crick (bottom) strand, and for bottom arrows, R-loop forms with the Watson (top) strand. (B) Cleavage reaction setup. nuc, nucleosome substrate. (C) Representative gels of target 4 cleavage by Cas12a. (D) Cleavage rates for the six DNA and nucleosome Cas12a targets at 25°C. Downward arrows signify that the value is an upper limit due to the lack of detectable cleavage. (E) Top: Diagram depicting variant 601 constructs (fig. S1E). Bottom: Cas12a cleavage of variant nucleosome substrates normalized to original 601 nucleosome substrate. (F) Top: Crystal structure of the 601 nucleosome [Protein Data Bank (PDB): 3LZ0 (80)] highlighting the amino acid modifications H3Y41E and H3K56Q, which mimic H3Y41ph and H3K56ac. Bottom: Cleavage rates of H3 mutant nucleosome normalized to the original wt nucleosome. n.d., no data, as no cleavage was observed for targets 3 and 4 for all nucleosome substrates. (D to F) Each data point is the mean of at least three replicates; error bars: SEM.

Fig. 2. Nucleosomes inhibit Cas12a’s two-step DNA binding.

(A) Concentration-dependent Cas12a cleavage plots of DNA (dark triangles) and nucleosome substrates (light circles). Colored lines: Hyperbolic curve fitting. Second-order rate constants for each target are written below. The asterisk indicates that the target 5 DNA cleavage second-order rate constant is a lower limit due to lack of data points in the concentration-dependent phase. Each data point is the mean of at least three replicates; error bars: SEM. (B) Dissociation rates determined by electrophoretic mobility shift assay (fig. S2). Data points represent at least duplicates; error bars: SEM. (C) Diagrams showing the calculated nucleosomal inhibition on Cas12a PAM recognition and R-loop formation (fig. S2C). K_PAM (Cas12a affinity for the targets’ PAM) increases and k_R-loop (rate of R-loop formation) decreases due to the nucleosome.

Fig. 3. Steric interference by a proximal nuclease enhances nucleosomal DNA cleavage.

(A) Diagram depicting dCas9 (light blue arrows) and Cas12a (color-coded arrows) targets. Only targets discussed in the main text are included here; others are shown in fig. S3. (B) Proxy-CRISPR reaction scheme. (C) Proxy-CRISPR nucleosome cleavage rates for indicated dCas9 and Cas12a pairs. n.a., original nucleosome cleavage rate without dCas9 prebound. Data points are normalized to the DNA cleavage rate of each target. Each data point is the mean of three replicates; error bars: SEM. (D) SpCas9 [PDB: 4UN3 (81)] modeled to bind target 5p, 13 bp away from the nucleosome [PDB: 3LZ0 (80)]. Linker DNA was generated using sequence to structure modeling. Extension of the distal linker DNA shows a steric clash with the bound Cas9.

Fig. 4. Phase-separated nucleosome arrays minimally inhibit DNA accessibility.

(A) Diagram of a 12-mer 601 array. Gray boxes: 601 positioning sequences; black lines: unique linker DNAs. Cas12a targets are highlighted (L4/5, magenta; Ex5, green; L8/9, cyan), and the associated arrows represent direction of R-loop formation. (B) Phase-separated nucleosome array droplets colocalize with dCas12a-L4/5. Histone H2B is labeled with AF594; L4/5 crRNA is labeled with AF488. Scale bar, 10 μm. (C) Reaction scheme depicting Cas12a cleavage of DNA arrays or phase-separated nucleosome arrays. (D) Representative agarose gel showing Cas12a cleavage of phase-separated nucleosome arrays, detected by ethidium bromide. All reactions are from the same gel; the black line represents cropping of the gel. (E) Cas12a cleavage plot of DNA and nucleosome arrays. Data points are color-coordinated with (A). Rates are included in table S5. (F) Concentration-dependent Cas12a cleavage of L4/5, targeting DNA, and nucleosome arrays. (E and F) Each data point represents the mean of at least three replicates (exception: DNA cleavage at 30 and 200 nM Cas12a was done in duplicate). Error bars: SEM. (G) Model of Cas12a cleavage inhibition by the nucleosome and chromatin. Chromatin has a small impact on Cas12a cleavage efficiency, which can be attributed to neighboring nucleosomes. The nucleosome inhibits Cas12a binding to wrapped DNA.