8W2S

Cas9d Effector:sgRNA Binary Complex

Summary for 8W2S

• Entry DOI: 10.2210/pdb8w2s/pdb
• EMDB information: 43757
• Descriptor: HNH nuclease domain-containing protein, sgRNA (2 entities in total)
• Functional Keywords: crispr-cas9, endonuclease, binary complex, effector, sgrna, immune system-rna complex, immune system/rna
• Biological source: Deltaproteobacteria; More
• Total number of polymer chains: 2
• Total formula weight: 159825.80

Authors

Fregoso Ocampo, R.,Bravo, J.P.K.,Taylor, D.W. (deposition date: 2024-02-21, release date: 2025-01-15, Last modification date: 2025-06-04)

Primary citation

Ocampo, R.F.,Bravo, J.P.K.,Dangerfield, T.L.,Nocedal, I.,Jirde, S.A.,Alexander, L.M.,Thomas, N.C.,Das, A.,Nielson, S.,Johnson, K.A.,Brown, C.T.,Butterfield, C.N.,Goltsman, D.S.A.,Taylor, D.W.
DNA targeting by compact Cas9d and its resurrected ancestor.
Nat Commun, 16:457-457, 2025

PubMed Abstract: 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.

PubMed: 39774105
DOI: 10.1038/s41467-024-55573-4

Experimental method

ELECTRON MICROSCOPY (3.4 Å)