6o0z › Citations

Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9.

<div class='caption-title'>Cryo-EM structures of three states of Cas9-sgRNA-dsDNA complex.</div>
<div class='caption-title'>The central channel of Cas9 accommodates the R-loop structure in state I.</div>
<div class='caption-title'>HNH domain adopts catalytic conformation in state II.</div>
Zhu X, Clarke R, Puppala AK, Chittori S, Merk A, Merrill BJ, Simonović M, Subramaniam S
OpenAccess logo Nat Struct Mol Biol 26 679-685 (2019)
Related entries: 6o0x, 6o0y

Cited: 61 times
EuropePMC logo PMID: 31285607

Abstract

The RNA-guided Cas9 endonuclease from Streptococcus pyogenes is a single-turnover enzyme that displays a stable product state after double-stranded-DNA cleavage. Here, we present cryo-EM structures of precatalytic, postcatalytic and product states of the active Cas9-sgRNA-DNA complex in the presence of Mg2+. In the precatalytic state, Cas9 adopts the 'checkpoint' conformation with the HNH nuclease domain positioned far away from the DNA. Transition to the postcatalytic state involves a dramatic ~34-Å swing of the HNH domain and disorder of the REC2 recognition domain. The postcatalytic state captures the cleaved substrate bound to the catalytically competent HNH active site. In the product state, the HNH domain is disordered, REC2 returns to the precatalytic conformation, and additional interactions of REC3 and RuvC with nucleic acids are formed. The coupled domain motions and interactions between the enzyme and the RNA-DNA hybrid provide new insights into the mechanism of genome editing by Cas9.

Reviews - 6o0z mentioned but not cited (2)

  1. Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes. Wang J, Arantes PR, Ahsan M, Sinha S, Kyro GW, Maschietto F, Allen B, Skeens E, Lisi GP, Batista VS, Palermo G. Front Mol Biosci 9 1072733 (2022)
  2. Insights into the Mechanism of CRISPR/Cas9-Based Genome Editing from Molecular Dynamics Simulations. Bhattacharya S, Satpati P. ACS Omega 8 1817-1837 (2023)

Articles - 6o0z mentioned but not cited (8)

  1. Cryo-EM structures reveal coordinated domain motions that govern DNA cleavage by Cas9. Zhu X, Clarke R, Puppala AK, Chittori S, Merk A, Merrill BJ, Simonović M, Subramaniam S. Nat Struct Mol Biol 26 679-685 (2019)
  2. R-loop formation and conformational activation mechanisms of Cas9. Pacesa M, Loeff L, Querques I, Muckenfuss LM, Sawicka M, Jinek M. Nature 609 191-196 (2022)
  3. Atomic-scale insights into allosteric inhibition and evolutional rescue mechanism of Streptococcus thermophilus Cas9 by the anti-CRISPR protein AcrIIA6. Li X, Wang C, Peng T, Chai Z, Ni D, Liu Y, Zhang J, Chen T, Lu S. Comput Struct Biotechnol J 19 6108-6124 (2021)
  4. CHARMM-GUI Drude prepper for molecular dynamics simulation using the classical Drude polarizable force field. Kognole AA, Lee J, Park SJ, Jo S, Chatterjee P, Lemkul JA, Huang J, MacKerell AD, Im W. J Comput Chem 43 359-375 (2022)
  5. Engineering multiple species-like genetic incompatibilities in insects. Maselko M, Feltman N, Upadhyay A, Hayward A, Das S, Myslicki N, Peterson AJ, O'Connor MB, Smanski MJ. Nat Commun 11 4468 (2020)
  6. Real-time observation of Cas9 postcatalytic domain motions. Wang Y, Mallon J, Wang H, Singh D, Hyun Jo M, Hua B, Bailey S, Ha T. Proc Natl Acad Sci U S A 118 e2010650118 (2021)
  7. research-article Application of monolayer graphene to cryo-electron microscopy grids for high-resolution structure determination. Grassetti AV, May MB, Davis JH. bioRxiv 2023.07.28.550908 (2023)
  8. Coevolutionary Couplings Unravel PAM-Proximal Constraints of CRISPR-SpCas9. Li Y, De la Paz JA, Jiang X, Liu R, Pokkulandra AP, Bleris L, Morcos F. Biophys J 117 1684-1691 (2019)


Reviews citing this publication (11)

  1. CRISPR/Cas9 therapeutics: progress and prospects. Li T, Yang Y, Qi H, Cui W, Zhang L, Fu X, He X, Liu M, Li PF, Yu T. Signal Transduct Target Ther 8 36 (2023)
  2. Challenges and Perspectives in Homology-Directed Gene Targeting in Monocot Plants. Van Vu T, Sung YW, Kim J, Doan DTH, Tran MT, Kim JY. Rice (N Y) 12 95 (2019)
  3. Structural biology of CRISPR-Cas immunity and genome editing enzymes. Wang JY, Pausch P, Doudna JA. Nat Rev Microbiol 20 641-656 (2022)
  4. Allosteric regulation of CRISPR-Cas9 for DNA-targeting and cleavage. Zuo Z, Liu J. Curr Opin Struct Biol 62 166-174 (2020)
  5. Target binding and residence: a new determinant of DNA double-strand break repair pathway choice in CRISPR/Cas9 genome editing. Feng Y, Liu S, Chen R, Xie A. J Zhejiang Univ Sci B 22 73-86 (2021)
  6. Targeting cancer epigenetics with CRISPR-dCAS9: Principles and prospects. Rahman MM, Tollefsbol TO. Methods 187 77-91 (2021)
  7. Dynamics and mechanisms of CRISPR-Cas9 through the lens of computational methods. Saha A, Arantes PR, Palermo G. Curr Opin Struct Biol 75 102400 (2022)
  8. Machines on Genes through the Computational Microscope. Sinha S, Pindi C, Ahsan M, Arantes PR, Palermo G. J Chem Theory Comput 19 1945-1964 (2023)
  9. CRISPR-Combo-mediated orthogonal genome editing and transcriptional activation for plant breeding. Pan C, Qi Y. Nat Protoc 18 1760-1794 (2023)
  10. Development of Single Molecule Techniques for Sensing and Manipulation of CRISPR and Polymerase Enzymes. Chu J, Romero A, Taulbee J, Aran K. Small 19 e2300328 (2023)
  11. Engineering Cas9: next generation of genomic editors. Kovalev MA, Davletshin AI, Karpov DS. Appl Microbiol Biotechnol 108 209 (2024)

Articles citing this publication (40)

  1. Structural basis for mismatch surveillance by CRISPR-Cas9. Bravo JPK, Liu MS, Hibshman GN, Dangerfield TL, Jung K, McCool RS, Johnson KA, Taylor DW. Nature 603 343-347 (2022)
  2. Gaussian accelerated molecular dynamics (GaMD): principles and applications. Wang J, Arantes PR, Bhattarai A, Hsu RV, Pawnikar S, Huang YM, Palermo G, Miao Y. Wiley Interdiscip Rev Comput Mol Sci 11 e1521 (2021)
  3. Structures of Neisseria meningitidis Cas9 Complexes in Catalytically Poised and Anti-CRISPR-Inhibited States. Sun W, Yang J, Cheng Z, Amrani N, Liu C, Wang K, Ibraheim R, Edraki A, Huang X, Wang M, Wang J, Liu L, Sheng G, Yang Y, Lou J, Sontheimer EJ, Wang Y. Mol Cell 76 938-952.e5 (2019)
  4. Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA. Liu MS, Gong S, Yu HH, Jung K, Johnson KA, Taylor DW. Nat Commun 11 3576 (2020)
  5. Structural basis for Cas9 off-target activity. Pacesa M, Lin CH, Cléry A, Saha A, Arantes PR, Bargsten K, Irby MJ, Allain FH, Palermo G, Cameron P, Donohoue PD, Jinek M. Cell 185 4067-4081.e21 (2022)
  6. Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a. Saha A, Arantes PR, Hsu RV, Narkhede YB, Jinek M, Palermo G. J Chem Inf Model 60 6427-6437 (2020)
  7. Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9. Sapozhnikov DM, Szyf M. Nat Commun 12 5711 (2021)
  8. Establishing the allosteric mechanism in CRISPR-Cas9. Nierzwicki Ł, Arantes PR, Saha A, Palermo G. Wiley Interdiscip Rev Comput Mol Sci 11 e1503 (2021)
  9. Structure of a type IV CRISPR-Cas ribonucleoprotein complex. Zhou Y, Bravo JPK, Taylor HN, Steens JA, Jackson RN, Staals RHJ, Taylor DW. iScience 24 102201 (2021)
  10. Quantification of Cas9 binding and cleavage across diverse guide sequences maps landscapes of target engagement. Boyle EA, Becker WR, Bai HB, Chen JS, Doudna JA, Greenleaf WJ. Sci Adv 7 eabe5496 (2021)
  11. Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR-Cas9. Nierzwicki Ł, East KW, Binz JM, Hsu RV, Ahsan M, Arantes PR, Skeens E, Pacesa M, Jinek M, Lisi GP, Palermo G. Nat Catal 5 912-922 (2022)
  12. Molecular Dynamics to Predict Cryo-EM: Capturing Transitions and Short-Lived Conformational States of Biomolecules. Nierzwicki Ł, Palermo G. Front Mol Biosci 8 641208 (2021)
  13. Prime editing with genuine Cas9 nickases minimizes unwanted indels. Lee J, Lim K, Kim A, Mok YG, Chung E, Cho SI, Lee JM, Kim JS. Nat Commun 14 1786 (2023)
  14. Active-Site Models of Streptococcus pyogenes Cas9 in DNA Cleavage State. Tang H, Yuan H, Du W, Li G, Xue D, Huang Q. Front Mol Biosci 8 653262 (2021)
  15. Structural Basis for Reduced Dynamics of Three Engineered HNH Endonuclease Lys-to-Ala Mutants for the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Associated 9 (CRISPR/Cas9) Enzyme. Wang J, Skeens E, Arantes PR, Maschietto F, Allen B, Kyro GW, Lisi GP, Palermo G, Batista VS. Biochemistry 61 785-794 (2022)
  16. Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR-Cas9 variants. Murugan K, Suresh SK, Seetharam AS, Severin AJ, Sashital DG. Nucleic Acids Res 49 4037-4053 (2021)
  17. Probing the stability of the SpCas9-DNA complex after cleavage. Aldag P, Welzel F, Jakob L, Schmidbauer A, Rutkauskas M, Fettes F, Grohmann D, Seidel R. Nucleic Acids Res 49 12411-12421 (2021)
  18. Structure and engineering of the minimal type VI CRISPR-Cas13bt3. Nakagawa R, Kannan S, Altae-Tran H, Takeda SN, Tomita A, Hirano H, Kusakizako T, Nishizawa T, Yamashita K, Zhang F, Nishimasu H, Nureki O. Mol Cell 82 3178-3192.e5 (2022)
  19. Terminal Uridylyl Transferase Mediated Site-Directed Access to Clickable Chromatin Employing CRISPR-dCas9. George JT, Azhar M, Aich M, Sinha D, Ambi UB, Maiti S, Chakraborty D, Srivatsan SG. J Am Chem Soc 142 13954-13965 (2020)
  20. Coordinated Actions of Cas9 HNH and RuvC Nuclease Domains Are Regulated by the Bridge Helix and the Target DNA Sequence. Babu K, Kathiresan V, Kumari P, Newsom S, Parameshwaran HP, Chen X, Liu J, Qin PZ, Rajan R. Biochemistry 60 3783-3800 (2021)
  21. Structural basis of Staphylococcus aureus Cas9 inhibition by AcrIIA14. Liu H, Zhu Y, Lu Z, Huang Z. Nucleic Acids Res 49 6587-6595 (2021)
  22. Engineering of the genome editing protein Cas9 to slide along DNA. Banerjee T, Takahashi H, Subekti DRG, Kamagata K. Sci Rep 11 14165 (2021)
  23. Improving the on-target activity of high-fidelity Cas9 editors by combining rational design and random mutagenesis. Spasskaya DS, Davletshin AI, Bachurin SS, Tutyaeva VV, Garbuz DG, Karpov DS. Appl Microbiol Biotechnol 107 2385-2401 (2023)
  24. Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects. Zuo Z, Babu K, Ganguly C, Zolekar A, Newsom S, Rajan R, Wang YC, Liu J. CRISPR J 5 329-340 (2022)
  25. Crystal structure of an RNA/DNA strand exchange junction. Cofsky JC, Knott GJ, Gee CL, Doudna JA. PLoS One 17 e0263547 (2022)
  26. Site-Specific Labeling Reveals Cas9 Induces Partial Unwinding Without RNA/DNA Pairing in Sequences Distal to the PAM. Li Y, Liu Y, Singh J, Tangprasertchai NS, Trivedi R, Fang Y, Qin PZ. CRISPR J 5 341-352 (2022)
  27. Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly. Sudhakar S, Barkau CL, Chilamkurthy R, Barber HM, Pater AA, Moran SD, Damha MJ, Pradeepkumar PI, Gagnon KT. J Biol Chem 299 104700 (2023)
  28. DNA rehybridization drives product release from Cas9 ribonucleoprotein to enable multiple-turnover cleavage. Pan J, Mabuchi M, Robb GB. Nucleic Acids Res 51 3903-3917 (2023)
  29. Genome-wide CRISPR off-target prediction and optimization using RNA-DNA interaction fingerprints. Chen Q, Chuai G, Zhang H, Tang J, Duan L, Guan H, Li W, Li W, Wen J, Zuo E, Zhang Q, Liu Q. Nat Commun 14 7521 (2023)
  30. HK97 gp74 Possesses an α-Helical Insertion in the ββα Fold That Affects Its Metal Binding, cos Site Digestion, and In Vivo Activities. Weiditch SA, Bickers SC, Bona D, Maxwell KL, Kanelis V. J Bacteriol 202 e00644-19 (2020)
  31. Increasing the Activity of the High-Fidelity SpyCas9 Form in Yeast by Directed Mutagenesis of the PAM-Interacting Domain. Davletshin AI, Matveeva AA, Bachurin SS, Karpov DS, Garbuz DG. Int J Mol Sci 25 444 (2023)
  32. The Electronic Structure of Genome Editors from the First Principles. Nierzwicki Ł, Ahsan M, Palermo G. Electron Struct 5 014003 (2023)
  33. Transcription-coupled donor DNA expression increases homologous recombination for efficient genome editing. Gao K, Zhang X, Zhang Z, Wu X, Guo Y, Fu P, Sun A, Peng J, Zheng J, Yu P, Wang T, Ye Q, Jiang J, Wang H, Lin CP, Gao G. Nucleic Acids Res 50 e109 (2022)
  34. A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules. Lim D, Zhou Q, Cox KJ, Law BK, Lee M, Kokkonda P, Sreekanth V, Pergu R, Chaudhary SK, Gangopadhyay SA, Maji B, Lai S, Amako Y, Thompson DB, Subramanian HKK, Mesleh MF, Dančík V, Clemons PA, Wagner BK, Woo CM, Church GM, Choudhary A. Nat Cell Biol 24 1766-1775 (2022)
  35. Acylation driven by intracellular metabolites in host cells inhibits Cas9 activity used for genome editing. Zhao L, You D, Wang T, Zou ZP, Yin BC, Zhou Y, Ye BC. PNAS Nexus 1 pgac277 (2022)
  36. Coupled catalytic states and the role of metal coordination in Cas9. Das A, Rai J, Roth MO, Shu Y, Medina ML, Barakat MR, Li H. Nat Catal 6 969-977 (2023)
  37. Deciphering the QR Code of the CRISPR-Cas9 System: Synergy between Gln768 (Q) and Arg976 (R). Daskalakis V. ACS Phys Chem Au 2 496-505 (2022)
  38. Guide-specific loss of efficiency and off-target reduction with Cas9 variants. Zhang L, He W, Fu R, Wang S, Chen Y, Xu H. Nucleic Acids Res 51 9880-9893 (2023)
  39. Structural insights into Cas9 mismatch: promising for development of high-fidelity Cas9 variants. Tang H, Wang D, Shu Y. Signal Transduct Target Ther 7 271 (2022)
  40. Substrate-independent activation pathways of the CRISPR-Cas9 HNH nuclease. Wang J, Maschietto F, Qiu T, Arantes PR, Skeens E, Palermo G, Lisi GP, Batista VS. Biophys J 122 4635-4644 (2023)


Quick links