Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA

Overview

Journal Cell

Publisher Cell Press

Specialty Cell Biology

Date 2014 Feb 18

PMID 24529477

Citations 870

Authors

Hiroshi Nishimasu

F Ann Ran

Patrick D Hsu

Silvana Konermann

Soraya I Shehata

Naoshi Dohmae

Ryuichiro Ishitani

Feng Zhang

Osamu Nureki

Affiliations

Soon will be listed here.

Abstract

The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.

Citing Articles

Insights into the compact CRISPR-Cas9d system.

Yang J, Wang T, Huang Y, Long Z, Li X, Zhang S Nat Commun. 2025; 16(1):2462.

PMID: 40075056 PMC: 11903963. DOI: 10.1038/s41467-025-57455-9.

CRISPR-Cas applications in agriculture and plant research.

Tuncel A, Pan C, Clem J, Liu D, Qi Y Nat Rev Mol Cell Biol. 2025; .

PMID: 40055491 DOI: 10.1038/s41580-025-00834-3.

CRISPR/Cas9-mediated efficient PlCYP81Q38 mutagenesis in Phryma leptostachya.

Pei Y, Cao W, Kong X, Wang S, Sun Z, Zuo Y Planta. 2025; 261(4):73.

PMID: 40029441 DOI: 10.1007/s00425-025-04657-6.

Improving adenine base editing precision by enlarging the recognition domain of CRISPR-Cas9.

Gao S, Weng B, Wich D, Power L, Chen M, Guan H Nat Commun. 2025; 16(1):2081.

PMID: 40021632 PMC: 11871365. DOI: 10.1038/s41467-025-57154-5.

Utilizing Target Sequences with Multiple Flanking Protospacer Adjacent Motif (PAM) Sites Reduces Off-Target Effects of the Cas9 Enzyme in Pineapple.

Shu H, Luan A, Ullah H, He J, Wang Y, Chen C Genes (Basel). 2025; 16(2).

PMID: 40004545 PMC: 11855603. DOI: 10.3390/genes16020217.

References

Spilman M, Cocozaki A, Hale C, Shao Y, Ramia N, Terns R . Structure of an RNA silencing complex of the CRISPR-Cas immune system. Mol Cell. 2013; 52(1):146-52. PMC: 3864027. DOI: 10.1016/j.molcel.2013.09.008. View

Gratz S, Cummings A, Nguyen J, Hamm D, Donohue L, Harrison M . Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease. Genetics. 2013; 194(4):1029-35. PMC: 3730909. DOI: 10.1534/genetics.113.152710. View

Mojica F, Diez-Villasenor C, Garcia-Martinez J, Almendros C . Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology (Reading). 2009; 155(Pt 3):733-740. DOI: 10.1099/mic.0.023960-0. View

Shalem O, Sanjana N, Hartenian E, Shi X, Scott D, Mikkelson T . Genome-scale CRISPR-Cas9 knockout screening in human cells. Science. 2013; 343(6166):84-87. PMC: 4089965. DOI: 10.1126/science.1247005. View

Wang H, Yang H, Shivalila C, Dawlaty M, Cheng A, Zhang F . One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013; 153(4):910-8. PMC: 3969854. DOI: 10.1016/j.cell.2013.04.025. View

Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S . CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007; 315(5819):1709-12. DOI: 10.1126/science.1138140. View

Horvath P, Barrangou R . CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010; 327(5962):167-70. DOI: 10.1126/science.1179555. View

Gorecka K, Komorowska W, Nowotny M . Crystal structure of RuvC resolvase in complex with Holliday junction substrate. Nucleic Acids Res. 2013; 41(21):9945-55. PMC: 3834835. DOI: 10.1093/nar/gkt769. View

Wiedenheft B, Lander G, Zhou K, Jore M, Brouns S, van der Oost J . Structures of the RNA-guided surveillance complex from a bacterial immune system. Nature. 2011; 477(7365):486-489. PMC: 4165517. DOI: 10.1038/nature10402. View

10.

Qi L, Larson M, Gilbert L, Doudna J, Weissman J, Arkin A . Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell. 2013; 152(5):1173-83. PMC: 3664290. DOI: 10.1016/j.cell.2013.02.022. View

11.

Yang H, Wang H, Shivalila C, Cheng A, Shi L, Jaenisch R . One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering. Cell. 2013; 154(6):1370-9. PMC: 3961003. DOI: 10.1016/j.cell.2013.08.022. View

12.

Fu Y, Foden J, Khayter C, Maeder M, Reyon D, Joung J . High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol. 2013; 31(9):822-6. PMC: 3773023. DOI: 10.1038/nbt.2623. View

13.

Cong L, Ran F, Cox D, Lin S, Barretto R, Habib N . Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339(6121):819-23. PMC: 3795411. DOI: 10.1126/science.1231143. View

14.

Terns M, Terns R . CRISPR-based adaptive immune systems. Curr Opin Microbiol. 2011; 14(3):321-7. PMC: 3119747. DOI: 10.1016/j.mib.2011.03.005. View

15.

Pattanayak V, Lin S, Guilinger J, Ma E, Doudna J, Liu D . High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity. Nat Biotechnol. 2013; 31(9):839-43. PMC: 3782611. DOI: 10.1038/nbt.2673. View

16.

Holm L, Rosenstrom P . Dali server: conservation mapping in 3D. Nucleic Acids Res. 2010; 38(Web Server issue):W545-9. PMC: 2896194. DOI: 10.1093/nar/gkq366. View

17.

Deveau H, Garneau J, Moineau S . CRISPR/Cas system and its role in phage-bacteria interactions. Annu Rev Microbiol. 2010; 64:475-93. DOI: 10.1146/annurev.micro.112408.134123. View

18.

Hwang W, Fu Y, Reyon D, Maeder M, Tsai S, Sander J . Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013; 31(3):227-9. PMC: 3686313. DOI: 10.1038/nbt.2501. View

19.

Maeder M, Linder S, Cascio V, Fu Y, Ho Q, Joung J . CRISPR RNA-guided activation of endogenous human genes. Nat Methods. 2013; 10(10):977-9. PMC: 3794058. DOI: 10.1038/nmeth.2598. View

20.

Biertumpfel C, Yang W, Suck D . Crystal structure of T4 endonuclease VII resolving a Holliday junction. Nature. 2007; 449(7162):616-20. DOI: 10.1038/nature06152. View