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Structure paper

TitleStructural basis for target DNA cleavage and guide RNA processing by CRISPR-Casλ2.
Journal, issue, pagesCommun Biol, Vol. 8, Issue 1, Page 876, Year 2025
Publish dateJun 5, 2025
AuthorsSatoshi N Omura / Lauren E Alfonse / Alexa Ornstein / Hayato Morinaga / Hisato Hirano / Yuzuru Itoh / Gabrielle Munoz / Anthony J Garrity / Gregory R Hoffman / Tia DiTommaso / Winston X Yan / David R Cheng / David A Scott / Zachary Maben / Osamu Nureki /
PubMed AbstractRNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ-also referred to as Cas12n-is a recently identified ...RNA-guided CRISPR-Cas nucleases are widely used as versatile genome-engineering tools. Among the diverse CRISPR-Cas effectors, CRISPR-Casλ-also referred to as Cas12n-is a recently identified miniature type V nuclease encoded in phage genomes. Given its demonstrated nuclease activity in both mammalian and plant cells, Casλ has emerged as a promising candidate for genome-editing applications. However, the precise molecular mechanisms of Casλ family enzymes remain poorly understood. In this study, we report the identification and detailed biochemical and structural characterizations of CRISPR-Casλ2. The cryo-electron microscopy structures of Casλ2 in five different functional states unveiled the dynamic domain rearrangements during its activation. Our biochemical analyses indicated that Casλ2 processes its precursor crRNA to a mature crRNA using the RuvC active site through a unique ruler mechanism, in which Casλ2 defines the spacer length of the mature crRNA. Furthermore, structural comparisons of Casλ2 with Casλ1 and CasΦ highlighted the diversity and conservation of phage-encoded type V CRISPR-Cas enzymes. Collectively, our findings augment the mechanistic understanding of diverse CRISPR-Cas nucleases and establish a framework for rational engineering of the CRISPR-Casλ-based genome-editing platform.
External linksCommun Biol / PubMed:40473912 / PubMed Central
MethodsEM (single particle)
Resolution2.84 - 3.06 Å
Structure data

61037

9izm

EMDB-61037, PDB-9izm:
Cryo-EM structure of CasLambda2-crRNA binary complex
Method: EM (single particle) / Resolution: 3.02 Å

61038

9izp

EMDB-61038, PDB-9izp:
Cryo-EM structure of CasLambda2-crRNA-target DNA ternary complex in the incompetent state
Method: EM (single particle) / Resolution: 2.89 Å

61039

9izq

EMDB-61039, PDB-9izq:
Cryo-EM structure of CasLambda2-crRNA-target DNA ternary complex in the intermediate state
Method: EM (single particle) / Resolution: 3.06 Å

61040

9izr

EMDB-61040, PDB-9izr:
Cryo-EM structure of CasLambda2-crRNA-target DNA ternary complex in the NTS-cleaving state
Method: EM (single particle) / Resolution: 2.93 Å

61041

9izs

EMDB-61041, PDB-9izs:
Cryo-EM structure of CasLambda2-crRNA-target DNA ternary complex in the TS-cleaving state
Method: EM (single particle) / Resolution: 2.84 Å

Chemicals

ChemComp-MG:
Unknown entry

ChemComp-GS:
GUANOSINE-5'-THIO-MONOPHOSPHATE

ChemComp-SC:
2-DEOXY-CYTIDINE-5'-THIOPHOSPHORATE

ChemComp-AS:
2-DEOXY-ADENOSINE -5'-THIO-MONOPHOSPHATE

Source
  • unidentified (others)
KeywordsDNA BINDING PROTEIN / CRISPR-CAS12 / GENOME ENGINEERING / HYDROLASE-RNA-DNA COMPLEX

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