8W2Z

Cas9d 6bp R-loop Seed Complex

8W2Z の概要

• エントリーDOI: 10.2210/pdb8w2z/pdb
• EMDBエントリー: 43760
• 分子名称: HNH nuclease domain-containing protein, sgRNA, DNA Target Strand, ... (4 entities in total)
• 機能のキーワード: crispr-cas9, endonuclease, binary complex, effector, sgrna, immune system-rna-dna complex, immune system/rna/dna
• 由来する生物種: Deltaproteobacteria; 詳細
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 158584.35

構造登録者

Fregoso Ocampo, R.,Bravo, J.P.K.,Taylor, D.W. (登録日: 2024-02-21, 公開日: 2025-01-15, 最終更新日: 2025-05-28)

主引用文献

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

実験手法

ELECTRON MICROSCOPY (3.37 Å)