Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2

Abstract

To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2–crRNA–DNA complexes and a Cas12i2–crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1–8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9–15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

Mechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1

“…Typically, the conserved RNase H fold utilizes a two-metal-ion catalytic mechanism for phosphoryl hydrolysis 40 , 41 . Considering that the RuvC domain in Cas12i1 also possesses such RNase H fold, positions of these water molecules in the present structure are possibly occupied by two-metal ions, which would be in accordance with the recently reported presence of two metal ions in the RuvC active site of the Cas12i2 ternary complex 28 .…”

“…The NTD strand was successively cleaved 13–15 nucleotides after the PAM duplex, and the TD strand was cleaved 24 nucleotides after the PAM duplex. Cas12i2 was also found to cleave the TD strand at the same position as observed in Cas12i1, whereas the NTD strand was primarily cleaved 31 nucleotides after the PAM duplex by Cas12i2 28 . In addition, the post-cleavage R-loop complex of Cas12i1 provides not only the information of the R-loop structure after the cis -cleavage of the target duplex, but also the information regarding the Cas12i1 trans -cleavage.…”

“…In the Cas12i1 pre-cleavage R-loop complex, the nucleotide of the TD strand swings to interact with that of the NTD strand beyond the 19-bp heteroduplex. In contrast, 28-bp and 26-bp heteroduplexes were previously reported to be accommodated in the Cas12i1 and Cas12i2 ternary complexes, respectively 27 , 28 , possibly due to the incomplete states of the target DNA duplexes and longer crRNA guide regions used to prepare the ternary complexes. This implies that the complete R-loop structure in Cas12i will affect the reasonable length of the heteroduplex formed by the TD strand and the crRNA guide region.…”

“…In the recently reported cryo-EM and crystal structures of Cas12i1 and Cas12i2 complexes, the partial target duplex containing the PAM sequences was presented 27 , 28 . The mechanism by which Cas12i unzips the double-stranded target duplex was short of investigation and information related to the R-loop formation and recognition in Cas12i was almost not reported.…”

“…More recently, cryo-EM and crystal structures of Cas12i in crRNA-bound and crRNA-target-bound states have been reported 27 , 28 . However, a number of key questions concerning the target DNA duplex unwinding, R-loop formation, target DNA duplex cleavage, key residues responsible for PAM determination in Cas12i1, and the mechanism of the DNase activation remain unanswered or require further investigation.…”