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

Martin Jínek; Krzysztof Chylinski; Ines Fonfara; M. Hauer; Jennifer A. Doudna

doi:10.1126/science.1225829

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

Martin Jínek, Krzysztof Chylinski, Ines Fonfara, M. Hauer, Jennifer A. Doudna, Emmanuelle Charpentier

American Association for the Advancement of Science (2012) • Volume 337, Issue 6096, Pages 816-821

Method-ToolMethod-ToolPDF AvailableGrade Eligible⚠️ Moderate Risk Flags

Overall Assessment

Adequate Methodological Quality

Assessment created by PaperScores Medical AI v0 on Dec 14, 2025

C

63/100

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Quality Dimensions

Bias & Integrity

COI handling, outcome switching, selective reporting

B-70

Methods Rigour

Protocol clarity, sampling, controls

B+80

External Validity

Generalisability, setting, population

C-55

Transparency & Reproducibility

Data/code/materials availability, preregistration

D-30

Significance & Novelty

Contribution vs prior art; context if known

A+98

Statistical Validity

Models, assumptions, multiplicity, CI priority

C60

Integrity & Transparency

Integrity checks

P-hacking risk?

?

None suspected

Signs of selective hypothesis testing or analysis choices that could inflate false positive rates.

Outcome switching?

?

None suspected

Evidence that primary outcomes were changed during or after the study, potentially distorting results.

Conflict of interest?

?

Review recommended

Authors have financial or other relationships that could bias research findings.

Data integrity issues?

?

None suspected

Concerns about the accuracy, completeness, or authenticity of the reported data.

Open science signals

Research data

?

Not shared

Raw data used in the analyses is publicly available or accessible upon request.

Analysis code

?

Not applicable

Code or scripts used to analyze the data are shared for reproducibility.

Study materials

?

Not shared

Protocols, questionnaires, and other materials are publicly available.

Premise

Primary Research Question

Does Cas9 use tracrRNA:crRNA to cleave dsDNA and can a single chimeric RNA program it?

Hypothesis

Cas9 is a dual-RNA-guided endonuclease recognising PAM and can be reprogrammed by sgRNA.

Hypothesis is Falsifiable

✓Yes

Defined cleavage assays and mutant domains provide clear tests.

PICO Framework

Population—Not specified

No human/animal population; in vitro enzyme studies.

Intervention—Not specified

No clinical intervention.

Comparison—Not specified

Comparators are molecular controls, not clinical.

Outcome—Not specified

Outcomes are cleavage/biochemical; not clinical.

Literature Positioning

Literature Review Balanced

Well covered

Background cites Type I/III systems and prior RNase III processing work.

Evidence: Intro; refs 1–38

Research Gap Clearly Stated

Well covered

Whether Cas9 directly cleaves DNA and RNA requirements unknown.

Evidence: Intro, p.1–2

Stated Contribution Clear

Well covered

Shows dual-RNA-guided Cas9 dsDNA cleavage and sgRNA engineering.

Evidence: Abstract; Conclusions

Study Provenance

Peer reviewed venueGrant funding disclosedConflict disclosure provided5 affiliations listed

Authors & Affiliations

UC Berkeley/HHMI; Umeå Univ (MIMS/UCMR); Univ Vienna (MFPL); LBNL; FMI Basel.

Funding Statement

HHMI, Austrian Science Fund, Univ Vienna, Swedish Research Council, Kempe Foundation, Umeå University.

Conflicts of Interest

Authors filed a related patent.

Peer Review

✓Science (2012) peer-reviewed article.

Evidence: Front matter

Methodological Assessment

All design modules are marked as not applicable. Use the accordion below to review their details.

Modules without applicable checklist items (9)

These designs were fully marked as not applicable but remain available for reference.

Abstract

Ditching Invading DNA Bacteria and archaea protect themselves from invasive foreign nucleic acids through an RNA-mediated adaptive immune system called CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated (Cas). Jinek et al. (p. 816 , published online 28 June; see the Perspective by Brouns ) found that for the type II CRISPR/Cas system, the CRISPR RNA (crRNA) as well as the trans-activating crRNA—which is known to be involved in the pre-crRNA processing—were both required to direct the Cas9 endonuclease to cleave the invading target DNA. Furthermore, engineered RNA molecules were able to program the Cas9 endonuclease to cleave specific DNA sequences to generate double-stranded DNA breaks.

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Study Overview

Study Design

Method Tool

Primary classification used to evaluate this research.

Population

In vitro biochemical assays using S. pyogenes Cas9 with tracrRNA:crRNA or chimeric sgRNA against plasmid/oligo dsDNA.

Brief summary of who or what was studied.

Pre-registration

Not registered

Indicates whether the protocol or trial was registered before data collection.

Reviewer Notes

Experimental enzyme/mechanism and tool creation; no participants or outcomes; fits Method-Tool.

Publication Details

DOI

10.1126/science.1225829

Published

June 29, 2012

Citations

16430 citations

FWCI

199.74

External Resources

DOI Link OpenAlex

Disclaimer: This assessment is generated by AI and should not be the sole basis for clinical or research decisions. Always review the original paper and consult with domain experts.

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