A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity

Journal: Science, published 29 Jun 2012
DOI: 10.1126/science.1225829
PubMed: PMID 22745249 · Cited in PubMed: 8280 articles

Citation figures are shown for reference only. They are not used to judge whether a study is standalone or a follow-up.

Assessment

Rule-based score: Unclear (follow-up index 50/100, see the score card)

Score card

Calculated by fixed rules from the data on this page. Each line shows what earned the points. Citation counts are not used.

Follow-up index

50/100

Unclear, following the group's own earlier work

55+ follow-up · 25 or less standalone · in between unclear

+7	Similar earlier article by the same group 10% keyword match: Small RNAs in streptococci (2012)
+15	Cites earlier studies by its own authors 8 studies
+17	Self-citations by ORCID iD 8 of 48 references (17%)
+0	Continuation wording none
+11	Very similar earlier article by another group 70% keyword match: STRUCTURE AND REACTIONS OF CLOSED DUPLEX DNA (1978)

Impact

27/100

Overall = 35% scientific advancement + 25% economic / commercial + 15% human resource development + 25% technologies & techniques

Scientific advancement35

mentions “reveal” · novelty (100 − follow-up index) × 0.3 = +15
Economic / commercial0

no matching terms
Human resource development21

no matching terms · 7 institutions, 6 authors = +21
Technologies & techniques45

3 × 15: CRISPR, CRISPR/Cas9, CRISPR and Genetic Engineering

Keywords used for the assessment (20)

System studied

Bacteriophages
RNA
Streptococcus pyogenes
Deoxyribonucleases, Type II Site-Specific
DNA Cleavage
DNA Breaks, Double-Stranded
Inverted Repeat Sequences
Trans-activating crRNA
Cas9
Endonuclease
DNA
Nucleic acid
Cleave

Technologies & techniques

CRISPR
CRISPR/Cas9
CRISPR and Genetic Engineering

Topic

sequence-specific DNA cleavage
DNA double-strand breaks
RNA regulation and disease
Bacterial Genetics and Biotechnology

Self-citations

8 of 48 references (17%) share an ORCID iD with this article's authors. 8 share an author by any match. 6 of 6 authors have an ORCID iD.

Jennifer A. Doudna: 6
Emmanuelle Charpentier: 2
Martin Jínek: 2
Krzysztof Chylinski: 1

Cited earlier studies by the same authors

Works in this article's reference list that share at least one author.

CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III (2011)
Shared authors: Krzysztof Chylinski, Emmanuelle Charpentier · DOI · PubMed
An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease Cse3 (2011)
Shared authors: Martin Jínek, Jennifer A. Doudna · DOI · PubMed
Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease (2010)
Shared authors: Martin Jínek, Jennifer A. Doudna · DOI · PubMed
RNA-guided genetic silencing systems in bacteria and archaea (2012)
Shared authors: Jennifer A. Doudna · DOI · PubMed
Mechanism of Foreign DNA Selection in a Bacterial Adaptive Immune System (2012)
Shared authors: Jennifer A. Doudna · DOI · PubMed
Evolution and classification of the CRISPR–Cas systems (2011)
Shared authors: Emmanuelle Charpentier · DOI · PubMed
RNA-guided complex from a bacterial immune system enhances target recognition through seed sequence interactions (2011)
Shared authors: Jennifer A. Doudna · DOI · PubMed
Structures of the RNA-guided surveillance complex from a bacterial immune system (2011)
Shared authors: Jennifer A. Doudna · DOI · PubMed

Authors

ORCID iDs as recorded in OpenAlex.

Martin Jínek ORCID 0000-0002-7601-210X
Howard Hughes Medical Institute; University of California, Berkeley
Krzysztof Chylinski ORCID 0000-0001-7029-8090
University of Vienna; Max Perutz Labs; Umeå Centre for Microbial Research
Ines Fonfara ORCID 0000-0003-4075-9217
Umeå Centre for Microbial Research; Umeå University
M. Hauer ORCID 0000-0001-7463-3191
University of California, Berkeley
Jennifer A. Doudna ORCID 0000-0001-9161-999X
Howard Hughes Medical Institute; Lawrence Berkeley National Laboratory; University of California, Berkeley
Emmanuelle Charpentier ORCID 0000-0002-0254-0778
Umeå Centre for Microbial Research; Umeå University

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

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