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TitleInnate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.
Journal, issue, pagesNucleic Acids Res, Vol. 52, Issue 6, Page 3234-3248, Year 2024
Publish dateApr 12, 2024
AuthorsGreta Bigelyte / Brigita Duchovska / Rimante Zedaveinyte / Giedrius Sasnauskas / Tomas Sinkunas / Indre Dalgediene / Giedre Tamulaitiene / Arunas Silanskas / Darius Kazlauskas / Lukas Valančauskas / Julene Madariaga-Marcos / Ralf Seidel / Virginijus Siksnys / Tautvydas Karvelis /
PubMed AbstractCas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) ...Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.
External linksNucleic Acids Res / PubMed:38261981 / PubMed Central
MethodsEM (single particle)
Resolution2.93 Å
Structure data

17757

8pm4

EMDB-17757, PDB-8pm4:
Cryo-EM structure of the Cas12m-crRNA-target DNA complex
Method: EM (single particle) / Resolution: 2.93 Å

Source
  • gordonia otitidis nbrc 100426 (bacteria)
  • synthetic construct (others)
KeywordsRNA BINDING PROTEIN / Cas12 / Cas12m / CRISPR-Cas / RuvC / crRNA / PAM

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