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TitleDNA targeting by compact Cas9d and its resurrected ancestor.
Journal, issue, pagesNat Commun, Vol. 16, Issue 1, Page 457, Year 2025
Publish dateJan 7, 2025
AuthorsRodrigo Fregoso Ocampo / Jack P K Bravo / Tyler L Dangerfield / Isabel Nocedal / Samatar A Jirde / Lisa M Alexander / Nicole C Thomas / Anjali Das / Sarah Nielson / Kenneth A Johnson / Christopher T Brown / Cristina N Butterfield / Daniela S A Goltsman / David W Taylor /
PubMed AbstractType II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). ...Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases.
External linksNat Commun / PubMed:39774105 / PubMed Central
MethodsEM (single particle)
Resolution2.73 - 3.4 Å
Structure data

43757

8w2s

EMDB-43757, PDB-8w2s:
Cas9d Effector:sgRNA Binary Complex
Method: EM (single particle) / Resolution: 3.4 Å

43760

8w2z

EMDB-43760, PDB-8w2z:
Cas9d 6bp R-loop Seed Complex
Method: EM (single particle) / Resolution: 3.37 Å

43878

9auf

EMDB-43878, PDB-9auf:
Cas9d 20bp R-loop Complex
Method: EM (single particle) / Resolution: 2.73 Å

Source
  • deltaproteobacteria (bacteria)
  • escherichia phage lambda (virus)
KeywordsIMMUNE SYSTEM/RNA / CRISPR-Cas9 / Endonuclease / Binary Complex / Effector / sgRNA / IMMUNE SYSTEM-RNA complex / IMMUNE SYSTEM/RNA/DNA / IMMUNE SYSTEM-RNA-DNA complex / DNA BINDING PROTEIN / RNA BINDING PROTEIN/DNA/RNA / DNA/RNA CRISPR Cas9 Cas9d Nuclease sgRNA / RNA BINDING PROTEIN-DNA-RNA complex

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