Skip to main content
. 2021 Mar 10;7(11):eabd6030. doi: 10.1126/sciadv.abd6030

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

Fig. 1

(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.