Structural basis for substrate recognition and cleavage by the dimerization-dependent CRISPR–Cas12f nuclease

Abstract

Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR–Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR–Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

Engineered miniature CRISPR-Cas system for mammalian genome regulation and editing

“…Interestingly, the single mutations at D143, T147, and E151 showing improved activity are near or in a (D/E)XRKN motif that is highly conserved in the Cas12a family (Figure 1J), suggesting that this domain could be important in regulating Cas12f-DNA interaction. Confirming this hypothesis, we aligned these mutations to very recently reported Cas12f structures and observed that they reside in the DNA binding pocket (Figures S4B and S4C;Takeda et al, 2021;Xiao et al, 2021). The variant D326A/D510A/D143R/T147R/K330R/E528R was chosen for further characterization for endogenous gene activation, which we refer to simply as ''dCasMINI'' hereafter.…”

Miniature type V-F CRISPR-Cas nucleases enable targeted DNA modification in cells

“…RNP complex stoichiometry. The cryo-electron microscopic (cryo-EM) structure of Un1Cas12f1 9,13 revealed that one gRNA and two Un1Cas12f1 nucleases comprise the RNP effector complex. Here, using mass photometry 14 , we evaluated the oligomeric state of SpCas12f1 and AsCas12f1 nucleases in the apo-form, in a binary complex with its gRNA and bound to a dsDNA target in a ternary complex (Fig.…”

Structural basis of target DNA recognition by CRISPR-Cas12k for RNA-guided DNA transposition

“…Although sharing low sequence similarity, REC1 13–102 is structurally similar to the REC1 C domain in Cas12f (Figs. 2D and S3B,C) , which forms the dimerization interface in Cas12f (Takeda et al, 2020; Xiao et al, 2021). However, the key hydrophobic residues (I118, Y121, Y122, I126) in Cas12f are not conserved in REC1 13–102 (Figs.…”

“…2A,B,E and S3B) . The Nuc domains or equivalent domains are inevitably present in all Cas12 proteins with structures determined to date including Cas12a (Dong et al, 2016; Gao et al, 2016; Nishimasu et al, 2017; Stella et al, 2017; Stella et al, 2018b; Swarts and Jinek, 2019; Swarts et al, 2017; Yamano et al, 2016; Yamano et al, 2017; Zhang et al, 2019), Cas12b (Liu et al, 2017; Wu et al, 2017; Yang et al, 2016), Cas12e (Liu et al, 2019), Cas12f (Takeda et al, 2020; Xiao et al, 2021), Cas12g (Li et al, 2021) and Cas12i (Huang et al, 2020; Zhang et al, 2020), and play an essential role in the nuclease activity. Second, the lid motif of Cas12k is longer than that of Cas12f and is in a closed conformation that covers the pseudonuclease site (Fig.…”

“…Cas12a (Dong et al, 2016; Gao et al, 2016; Nishimasu et al, 2017; Stella et al, 2017; Stella et al, 2018b; Swarts and Jinek, 2019; Swarts et al, 2017; Yamano et al, 2016; Yamano et al, 2017; Zhang et al, 2019), Cas12b (Liu et al, 2017; Wu et al, 2017; Yang et al, 2016), Cas12e (Liu et al, 2019) and Cas12i (Huang et al, 2020; Zhang et al, 2020)) or a second protein subunit (e.g. Cas12f (Takeda et al, 2020; Xiao et al, 2021)). The flexible REC1 103–270 might provide additional interactions with the heteroduplex.…”