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

A. Executive scientific assessment

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.

The AI assessment, research themes and proposals are not available for this map. The verified citation data below are complete.

B. Dynamic citation network

Arrows point from each paper to the work it builds on, labelled with the relationship read from its citing sentence. Grey dotted arrows mean the paper cites the original but its citing sentence was not available, so no stronger claim is made. Drag to pan, scroll to zoom, click a paper for details.

C. Chronological research timeline

Year → research group → publication → finding or relationship → influence within this map. New group marks a group's first paper here.

D. Research group influence map

Groups are identified by the corresponding (or last) author of each citing paper in this map. Two groups are shown as collaborating only when their papers share an author.

E. Research opportunity map

Research opportunities are written by AI and are not available for this map.

The 15 most-cited studies the original builds on, and 60 of its 17,118 citing papers (the most cited and the most recent). 9 have a citing sentence; the rest are marked “Cites (sentence not available)”.

G. Research strategy report

Research proposals are written by AI and are not available for this map.

Quantitative research impact

Total citations

17,118

Verified, OpenAlex

Citing papers in this map

60

Most cited + most recent

With citing sentence

9

Semantic Scholar, Europe PMC

Distinct research groups

44

In this map

Self-citations

7

Citing papers sharing an author

Most influential follow-up publications

Ranked by how many other papers in this map cite them, then by total citations. Citation counts measure attention, not validation.

1. Le Cong (2013). Multiplex Genome Engineering Using CRISPR/Cas Systems. 15,342 citations; cited by 34 in this map.
2. Prashant Mali (2013). RNA-Guided Human Genome Engineering via Cas9. 9,213 citations; cited by 30 in this map.
3. Patrick D. Hsu (2013). DNA targeting specificity of RNA-guided Cas9 nucleases. 4,741 citations; cited by 22 in this map.
4. Yanfang Fu (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. 3,294 citations; cited by 17 in this map.
5. Martin Jínek (2013). RNA-programmed genome editing in human cells. 2,098 citations; cited by 16 in this map.

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