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DNA interrogation by the CRISPR RNA-guided endonuclease Cas9

Author

Listed:
  • Samuel H. Sternberg

    (University of California)

  • Sy Redding

    (Columbia University)

  • Martin Jinek

    (Howard Hughes Medical Institute, University of California
    Present address: Department of Biochemistry, University of Zurich, 8057 Zurich, Switzerland.)

  • Eric C. Greene

    (Columbia University)

  • Jennifer A. Doudna

    (University of California
    Howard Hughes Medical Institute, University of California
    University of California
    Lawrence Berkeley National Laboratory)

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA–DNA base-pairing to target foreign DNA in bacteria. Cas9–guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9–RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9–RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9–RNA. Competition assays provide evidence that DNA strand separation and RNA–DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

Suggested Citation

  • Samuel H. Sternberg & Sy Redding & Martin Jinek & Eric C. Greene & Jennifer A. Doudna, 2014. "DNA interrogation by the CRISPR RNA-guided endonuclease Cas9," Nature, Nature, vol. 507(7490), pages 62-67, March.
  • Handle: RePEc:nat:nature:v:507:y:2014:i:7490:d:10.1038_nature13011
    DOI: 10.1038/nature13011
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    Cited by:

    1. Xu Feng & Ruyi Xu & Jianglan Liao & Jingyu Zhao & Baochang Zhang & Xiaoxiao Xu & Pengpeng Zhao & Xiaoning Wang & Jianyun Yao & Pengxia Wang & Xiaoxue Wang & Wenyuan Han & Qunxin She, 2024. "Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope," Nature Communications, Nature, vol. 15(1), pages 1-14, December.
    2. Lin Zhao & Sabrina R. T. Koseki & Rachel A. Silverstein & Nadia Amrani & Christina Peng & Christian Kramme & Natasha Savic & Martin Pacesa & Tomás C. Rodríguez & Teodora Stan & Emma Tysinger & Lauren , 2023. "PAM-flexible genome editing with an engineered chimeric Cas9," Nature Communications, Nature, vol. 14(1), pages 1-8, December.
    3. Sundaram Acharya & Asgar Hussain Ansari & Prosad Kumar Das & Seiichi Hirano & Meghali Aich & Riya Rauthan & Sudipta Mahato & Savitri Maddileti & Sajal Sarkar & Manoj Kumar & Rhythm Phutela & Sneha Gul, 2024. "PAM-flexible Engineered FnCas9 variants for robust and ultra-precise genome editing and diagnostics," Nature Communications, Nature, vol. 15(1), pages 1-23, December.
    4. Afsaneh Sadremomtaz & Robert F. Glass & Jorge Eduardo Guerrero & Dennis R. LaJeunesse & Eric A. Josephs & Reza Zadegan, 2023. "Digital data storage on DNA tape using CRISPR base editors," Nature Communications, Nature, vol. 14(1), pages 1-10, December.
    5. Jian Wang & Ke Wang & Zhe Deng & Zhiyu Zhong & Guo Sun & Qing Mei & Fuling Zhou & Zixin Deng & Yuhui Sun, 2024. "Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces," Nature Communications, Nature, vol. 15(1), pages 1-16, December.
    6. Adeeb Rahman & Neeti Sanan-Mishra, 2024. "When an Intruder Comes Home: GM and GE Strategies to Combat Virus Infection in Plants," Agriculture, MDPI, vol. 14(2), pages 1-26, February.
    7. Burcu Bestas & Sandra Wimberger & Dmitrii Degtev & Alexandra Madsen & Antje K. Rottner & Fredrik Karlsson & Sergey Naumenko & Megan Callahan & Julia Liz Touza & Margherita Francescatto & Carl Ivar Möl, 2023. "A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
    8. Yang Liu & Filipe Pinto & Xinyi Wan & Zhugen Yang & Shuguang Peng & Mengxi Li & Jonathan M. Cooper & Zhen Xie & Christopher E. French & Baojun Wang, 2022. "Reprogrammed tracrRNAs enable repurposing of RNAs as crRNAs and sequence-specific RNA biosensors," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    9. Dmitrii Degtev & Jack Bravo & Aikaterini Emmanouilidi & Aleksandar Zdravković & Oi Kuan Choong & Julia Liz Touza & Niklas Selfjord & Isabel Weisheit & Margherita Francescatto & Pinar Akcakaya & Michel, 2024. "Engineered PsCas9 enables therapeutic genome editing in mouse liver with lipid nanoparticles," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
    10. Aron Ferenczi & Yen Peng Chew & Erika Kroll & Charlotte Koppenfels & Andrew Hudson & Attila Molnar, 2021. "Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii," Nature Communications, Nature, vol. 12(1), pages 1-12, December.
    11. Dixit, Yatika & Yadav, Preeti & Sharma, Arun Kumar & Pandey, Poornima & Kuila, Arindam, 2023. "Multiplex genome editing to construct cellulase engineered Saccharomyces cerevisiae for ethanol production from cellulosic biomass," Renewable and Sustainable Energy Reviews, Elsevier, vol. 187(C).
    12. Shinta Saito & Noritaka Adachi, 2024. "Characterization and regulation of cell cycle-independent noncanonical gene targeting," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    13. Yi-Li Feng & Qian Liu & Ruo-Dan Chen & Si-Cheng Liu & Zhi-Cheng Huang & Kun-Ming Liu & Xiao-Ying Yang & An-Yong Xie, 2022. "DNA nicks induce mutational signatures associated with BRCA1 deficiency," Nature Communications, Nature, vol. 13(1), pages 1-15, December.
    14. Yanbo Wang & W. Taylor Cottle & Haobo Wang & Momcilo Gavrilov & Roger S. Zou & Minh-Tam Pham & Srinivasan Yegnasubramanian & Scott Bailey & Taekjip Ha, 2022. "Achieving single nucleotide sensitivity in direct hybridization genome imaging," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    15. Michael Kosicki & Felicity Allen & Frances Steward & Kärt Tomberg & Yangyang Pan & Allan Bradley, 2022. "Cas9-induced large deletions and small indels are controlled in a convergent fashion," Nature Communications, Nature, vol. 13(1), pages 1-11, December.
    16. András Tálas & Dorottya A. Simon & Péter I. Kulcsár & Éva Varga & Sarah L. Krausz & Ervin Welker, 2021. "BEAR reveals that increased fidelity variants can successfully reduce the mismatch tolerance of adenine but not cytosine base editors," Nature Communications, Nature, vol. 12(1), pages 1-14, December.
    17. Duško Lainšček & Vida Forstnerič & Veronika Mikolič & Špela Malenšek & Peter Pečan & Mojca Benčina & Matjaž Sever & Helena Podgornik & Roman Jerala, 2022. "Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing," Nature Communications, Nature, vol. 13(1), pages 1-12, December.

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