CRISPR-Cas12a exploits R-loop asymmetry to form double-strand breaks

Abstract

1Most type V CRISPR-Cas interference proteins use a single RuvC active site to make 2 RNA-guided breaks in double-stranded DNA substrates, an activity essential for both 3 bacterial immunity and genome editing applications. The best-studied of these 4 enzymes, Cas12a, initiates DNA cutting by forming a 20-nucleotide R-loop in which the 5 guide RNA displaces one of the DNA strands of a double-helical substrate, positioning 6 the DNase active site for first-strand cleavage. However, crystal structures and 7 biochemical data have not explained how the second strand is cut to complete the 8 double-strand break. Here, we show that Cas12a-mediated R-loop formation 9 destabilizes DNA at the second-strand cleavage site, which is located outside of the R-10 loop structure and beyond the 3′ end of the guide RNA. Chemical and fluorescent DNA 11 probes reveal that this destabilization is an intrinsic feature of DNA flanking the RNA-3′ 12 side of R-loops and does not require direct protein interactions. Interestingly, DNA 13 flanking the RNA-5′ side of R-loops is not intrinsically unstable. This asymmetry in R-14 loop structure may explain the uniformity of guide RNA architecture and the single-15 active-site cleavage mechanism that are fundamental features of all type V CRISPR-16Cas systems. 17 family occurs with opposing directionality (Fig.

CRISPR-Cas9 bends and twists DNA to read its sequence

“…5a). As anticipated for protein-induced base unstacking 24,25 , we detected a PAM-and Cas9-dependent increase in permanganate reactivity at Thy(+1) and Thy(+2) (Fig. 5a-c, Extended Data Fig.…”

“…After 2 min, 25 µL 2X stop solution (2 M βME, 30 mM EDTA) was added to stop the reaction, and 50 µL of water was added to each quenched reaction. The remainder of the protocol was conducted as described previously 25 , except an additional wash with 500 µL 70% ethanol was added to decrease the salt concentration in the final samples. Data analysis was similar to that described previously 25 .…”

“…The remainder of the protocol was conducted as described previously 25 , except an additional wash with 500 µL 70% ethanol was added to decrease the salt concentration in the final samples. Data analysis was similar to that described previously 25 . Let 𝑣 !…”

“…Duplex DNA substrates were annealed by heating to 95°C and cooling to 25°C over the course of 40 min on a thermocycler. Guide RNAs were transcribed and purified as described previously 25 , except no ribozyme was included in the transcript. All sgRNA molecules were annealed (80°C for 2 min, then moved directly to ice) in RNA storage buffer (0.1 mM EDTA, 2 mM sodium citrate, pH 6.4) prior to use.…”

“…Standard 5′ radiolabeling of DNA oligonucleotides was performed as described previously 25 . For 5′ radiolabeling of sgRNAs, the 5′ triphosphate was first removed by treatment with Quick CIP (New England BioLabs, manufacturer's instructions).…”