Evidence and sources

Measurements

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Big picture numbers

Structures

• 4CMP
  Apo SpCas9, no guide RNA

  The starting state of the cycle. Without guide RNA the two lobes sit in the wrong place, and this entry proves it. X-ray diffraction, 2.62 Å.
• 4CMQ
  Apo SpCas9 with manganese

  Companion to 4CMP. Use it to place the catalytic metal sites before any nucleic acid is present. X-ray diffraction, 3.09 Å.
• 4ZT0
  SpCas9 armed with single-guide RNA

  The hero of the scene. The guide RNA is loaded, the lobes have closed into a channel, and the enzyme is ready but has no DNA yet. X-ray diffraction, 2.9 Å.
• 4ZT9
  dCas9 with single-guide RNA

  The dCas9 swap with its own structure. Measured in this work: chain A superposes on 4ZT0 chain A at 1.13 A CA rmsd over 1312 residues. Killing catalysis leaves the machine intact. X-ray diffraction, 3.1 Å.
• 4UN3
  Cas9 bound to PAM-containing DNA

  Shows PAM readout by the PI domain, the step that licenses everything downstream. X-ray diffraction, 2.593 Å.
• 4OO8
  Ternary complex with guide RNA and target DNA

  The classic ternary structure that defined the bilobed architecture and placed HNH and RuvC against their strands. X-ray diffraction, 2.5 Å.
• 5F9R
  R-loop complex primed for cleavage

  The full R-loop with both DNA strands, caught with RuvC already on the non-target strand. The best single model of the pre-cleavage state. X-ray diffraction, 3.4 Å.
• 6O0Z
  Cryo-EM ternary complex, DNA still intact

  The before frame of the cleavage step: substrate intact, HNH present but far from the scissile strand. Electron microscopy, 3.3 Å.
• 6O0Y
  Cryo-EM complex with HNH docked and DNA cut

  The after frame. The target strand is in two pieces and HNH has docked onto the cut. This is the state that makes the cleavage step visible. Electron microscopy, 3.37 Å.
• 6O0X
  Cryo-EM complex with HNH not resolved

  Evidence that HNH is mobile rather than fixed. Do not morph to this entry: the domain the animation needs is missing from the model. Electron microscopy, 3.28 Å.
• 5Y36
  Cryo-EM ternary complex of dCas9

  An early cryo-EM view of the same complex. At 5.2 A it suits a low-detail overview shot only. RCSB gives no DOI for its primary citation. Electron microscopy, 5.2 Å.
• 6VPC
  ABE8e adenine base editor on DNA

  Shows the payload architecture: the same targeting module, with a chemistry module bolted on and aimed at the displaced strand. Electron microscopy, 3.2 Å.
• 5FW1
  SpCas9-VQR with a TGAG PAM

  The PAM swap with structural evidence. Same fold, different address alphabet. X-ray diffraction, 2.499 Å.
• 5B43
  Cas12a with crRNA and target DNA

  The side-by-side comparison. One guide RNA instead of two, a T-rich PAM, and a much shorter guide. X-ray diffraction, 2.8 Å.

References

1. 1UniProt Q99ZW2 (CAS9_STRP1), Streptococcus pyogenes serotype M1. 1368 residues, 158441 Da. Domain and active-site annotation: RuvC-I 1-62, bridge helix 56-73, recognition lobe 56-718, RuvC-II 718-765, HNH 770-921, RuvC-III 925-1102, PI 1099-1368, PAM substrate-binding motif 1333-1335, active sites Asp10 and His840
2. 2InterPro entries for UniProt Q99ZW2. Cas9-type HNH domain 770-921 (IPR033114), Cas9 RuvC domain 925-1099 (IPR055228), PAM-interacting domain 1102-1358 (IPR032237), REC lobe 181-712 (IPR032240), bridge helix 62-94 (IPR032239), RNase H-like superfamily 1-117 (CATH-Gene3D G3DSA:3.30.420.10)
3. 3Jinek et al., Science 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity
4. 4Jinek et al., Science 2014. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation
5. 5Nishimasu et al., Cell 2014. Crystal structure of Cas9 in complex with guide RNA and target DNA
6. 6Anders et al., Nature 2014. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease
7. 7Jiang et al., Science 2015. A Cas9-guide RNA complex preorganized for target DNA recognition
8. 8Jiang et al., Science 2016. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage
9. 9Sternberg et al., Nature 2014. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9
10. 10Sternberg et al., Nature 2015. Conformational control of DNA target cleavage by CRISPR-Cas9
11. 11Zhu et al., Nat Struct Mol Biol 2019. Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9
12. 12Komor et al., Nature 2016. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage
13. 13Gaudelli et al., Nature 2017. Programmable base editing of A.T to G.C in genomic DNA without DNA cleavage
14. 14Anzalone et al., Nature 2019. Search-and-replace genome editing without double-strand breaks or donor DNA
15. 15Kleinstiver et al., Nature 2016. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects
16. 16Slaymaker et al., Science 2016. Rationally engineered Cas9 nucleases with improved specificity
17. 17Chen et al., Nature 2017. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy
18. 18Nishimasu et al., Science 2018. Engineered CRISPR-Cas9 nuclease with expanded targeting space
19. 19Walton et al., Science 2020. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants
20. 20Lapinaite et al., Science 2020. DNA capture by a CRISPR-Cas9-guided adenine base editor
21. 21Anders et al., Mol Cell 2016. Structural plasticity of PAM recognition by engineered variants of the RNA-guided endonuclease Cas9
22. 22Zetsche et al., Cell 2015. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system
23. 23Yamano et al., Cell 2016. Crystal structure of Cpf1 in complex with guide RNA and target DNA