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TitleStructure of the type V-C CRISPR-Cas effector enzyme.
Journal, issue, pagesMol Cell, Vol. 82, Issue 10, Page 1865-11877.e4, Year 2022
Publish dateMay 19, 2022
AuthorsNina Kurihara / Ryoya Nakagawa / Hisato Hirano / Sae Okazaki / Atsuhiro Tomita / Kan Kobayashi / Tsukasa Kusakizako / Tomohiro Nishizawa / Keitaro Yamashita / David A Scott / Hiroshi Nishimasu / Osamu Nureki /
PubMed AbstractRNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR ...RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Recent studies identified functionally divergent type V Cas12 family enzymes. Among them, Cas12c2 binds a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) and recognizes double-stranded DNA targets with a short TN PAM. Here, we report the cryo-electron microscopy structures of the Cas12c2-guide RNA binary complex and the Cas12c2-guide RNA-target DNA ternary complex. The structures revealed that the crRNA and tracrRNA form an unexpected X-junction architecture, and that Cas12c2 recognizes a single T nucleotide in the PAM through specific hydrogen-bonding interactions with two arginine residues. Furthermore, our biochemical analyses indicated that Cas12c2 processes its precursor crRNA to a mature crRNA using the RuvC catalytic site through a unique mechanism. Collectively, our findings improve the mechanistic understanding of diverse type V CRISPR-Cas effectors.
External linksMol Cell / PubMed:35366394 / PubMed Central
MethodsEM (single particle)
Resolution2.7 - 3.0 Å
Structure data

31807

7v93

EMDB-31807, PDB-7v93:
Cryo-EM structure of the Cas12c2-sgRNA binary complex
Method: EM (single particle) / Resolution: 3.0 Å

31808

7v94

EMDB-31808, PDB-7v94:
Cryo-EM structure of the Cas12c2-sgRNA-target DNA ternary complex
Method: EM (single particle) / Resolution: 2.7 Å

Source
  • uncultured archaeon (environmental samples)
  • synthetic construct (others)
KeywordsRNA BINDING PROTEIN/RNA / cas12c / c2c3 / sgRNA / CRISPR / RNA binding protein-RNA complex / RNA BINDING PROTEIN/RNA/DNA / target DNA / RNA binding protein-RNA-DNA complex

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