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TitleDiscovery and structural mechanism of DNA endonucleases guided by RAGATH-18-derived RNAs.
Journal, issue, pagesCell Res, Year 2024
Publish dateApr 4, 2024
AuthorsKuan Ren / Fengxia Zhou / Fan Zhang / Mingyu Yin / Yuwei Zhu / Shouyu Wang / Yan Chen / Tengjin Huang / Zixuan Wu / Jiale He / Anqi Zhang / Changyou Guo / Zhiwei Huang /
PubMed AbstractCRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here ...CRISPR-Cas systems and IS200/IS605 transposon-associated TnpBs have been utilized for the development of genome editing technologies. Using bioinformatics analysis and biochemical experiments, here we present a new family of RNA-guided DNA endonucleases. Our bioinformatics analysis initially identifies the stable co-occurrence of conserved RAGATH-18-derived RNAs (reRNAs) and their upstream IS607 TnpBs with an average length of 390 amino acids. IS607 TnpBs form programmable DNases through interaction with reRNAs. We discover the robust dsDNA interference activity of IS607 TnpB systems in bacteria and human cells. Further characterization of the Firmicutes bacteria IS607 TnpB system (ISFba1 TnpB) reveals that its dsDNA cleavage activity is remarkably sensitive to single mismatches between the guide and target sequences in human cells. Our findings demonstrate that a length of 20 nt in the guide sequence of reRNA achieves the highest DNA cleavage activity for ISFba1 TnpB. A cryo-EM structure of the ISFba1 TnpB effector protein bound by its cognate RAGATH-18 motif-containing reRNA and a dsDNA target reveals the mechanisms underlying reRNA recognition by ISFba1 TnpB, reRNA-guided dsDNA targeting, and the sensitivity of the ISFba1 TnpB system to base mismatches between the guide and target DNA. Collectively, this study identifies the IS607 TnpB family of compact and specific RNA-guided DNases with great potential for application in gene editing.
External linksCell Res / PubMed:38575718
MethodsEM (single particle)
Resolution3.0 Å
Structure data

35323

8iaz

EMDB-35323, PDB-8iaz:
Cryo-EM structure of the ISFba1 TnpB-reRNA-dsDNA complex
Method: EM (single particle) / Resolution: 3.0 Å

Chemicals

ChemComp-ZN:
Unknown entry

Source
  • firmicutes bacterium am43-11bh (bacteria)
KeywordsRNA BINDING PROTEIN/RNA/DNA / RNA / Complex / RNA BINDING PROTEIN/RNA / RNA BINDING PROTEIN-RNA-DNA complex

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