Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria
Open accessAbstract
Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide adaptive immunity against viruses and plasmids in bacteria and archaea. The silencing of invading nucleic acids is executed by ribonucleoprotein complexes preloaded with small, interfering CRISPR RNAs (crRNAs) that act as guides for targeting and degradation of foreign nucleic acid. Here, we demonstrate that the Cas9–crRNA complex of the Streptococcus thermophilus CRISPR3/Cas system introduces in vitro a double-strand break at a specific site in DNA containing a sequence complementary to crRNA. DNA cleavage is executed by Cas9, which uses two distinct active sites, RuvC and HNH, to generate site-specific nicks on opposite DNA strands. Results demonstrate that the Cas9–crRNA complex functions as an RNA-guided endonuclease with RNA-directed target sequence recognition and protein-mediated DNA cleavage. These findings pave the way for engineering of universal programmable RNA-guided DNA endonucleases.
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DNA binding occurs at a 20-base pair DNA sequence complementary to a 20-nucleotide sequence in the guide RNA and can be readily altered by the experimenter.
“Importantly, DNA binding occurs at a 20-base pair DNA sequence that is complementary to a 20nucleotide sequence in the guide RNA and can be readily altered by the experimenter 1,20 (Fig. 2).”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [20]
The section “Cas9-crRNA Cleavage Specificity Is Directed by the crRNA Sequence” reports that 20 nucleotides of mature crRNA complement the protospacer and demonstrates that changing the spacer redirects targeting (page unavailable). But the Discussion states that double-stranded DNA binding requires both crRNA complementarity and an adjacent PAM; the claim omits that necessary condition. (LLM, conf=0.86)
Experimental — accuracy not yet measuredMutants lacking one of the Cas9 motifs become nicking endonucleases.
“Interestingly, mutants lacking one of the motifs become nicking endonucleases 16 .”
From Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus (2013) · cites this paper as [16]
The DNA recognition site must be adjacent to a PAM which triggers Cas9 to make a double-stranded DNA break within the targeted sequence.
“The DNA recognition site must be adjacent to a short motif (protospacer adjacent motif, PAM) which acts as a switch, triggering Cas9 to make a double-stranded DNA break within the targeted sequence 1,20 .”
From The promise and challenge of therapeutic genome editing (2020) · cites this paper as [20]
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“AHR mutations induced by gene editing Within the past decade, the generation of model systems has been aided by the advancement of CRISPRbased gene editing techniques (Gasiunas et al. 2012; Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013).”
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- Authors
- Gasiunas, G., Barrangou, R., Horvath, P., & Siksnys, V.
- Affiliations
- Institute of Biotechnology, Vilnius University, LT-02241 Vilnius, Lithuania;
- DuPont (United States) (US)
- DuPont (France) (FR)
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2012-09-04
- Volume
- 109
- Issue
- 39
- Pages
- E2579-86
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC3465414
- OpenAlex
- W2019670881
- Cited by
- 2,769
- References
- 27
- Citations by year
- 2026: 85 · 2025: 174 · 2024: 203 · 2023: 272 · 2022: 252 · 2021: 299 · 2020: 256 · 2019: 249 · 2018: 211 · 2017: 213 · 2016: 189 · 2015: 156 · 2014: 119 · 2013: 82 · 2012: 4
- FWCI
- 46.38
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- European Social Fund
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
- Keywords
- Cas9, crRNA, CRISPR-Cas, Streptococcus thermophilus, bacterial adaptive immunity, RNA-guided DNA endonuclease, DNA double-strand breaks, site-specific DNA cleavage, programmable nucleases
- MeSH terms
- Bacterial Proteins, Endonucleases, Ribonucleoproteins, RNA, Bacterial, Streptococcus thermophilus, DNA Breaks, Double-Stranded, DNA Breaks, Single-Stranded
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