A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity¶
Why this mattered¶
This paper transformed CRISPR-Cas9 from a bacterial immune mechanism into a programmable molecular tool. Its decisive contribution was to show that Cas9 could be directed to cut DNA at chosen sequences by RNA base pairing, and that the natural two-RNA guide system could be simplified into a single chimeric guide RNA. That simplification made the system legible as an engineering platform: changing the guide sequence, rather than redesigning a protein, could retarget DNA cleavage.
The paradigm shift was practical as much as conceptual. Earlier genome-editing tools such as zinc-finger nucleases and TALENs required custom protein engineering for each target site; this paper showed a route to genome targeting by Watson-Crick complementarity. After it, site-specific double-strand breaks became far easier to design, multiplex, and distribute across laboratories. The paper did not itself demonstrate editing in eukaryotic cells, but it supplied the biochemical mechanism and programmable architecture that made the 2013 demonstrations of CRISPR-Cas9 genome editing in mammalian and other cells possible.
Subsequent breakthroughs built directly on this programmable nuclease logic: genome-wide knockout screens, rapid model-organism engineering, somatic and ex vivo therapeutic editing, catalytically dead Cas9 for transcriptional control and imaging, and later precision-editing systems such as base editors and prime editors. Its importance lies in converting a natural defense system into a general-purpose platform for reading, perturbing, and rewriting genomes, thereby changing genome engineering from a specialist craft into a broadly accessible experimental method.
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.
Related¶
- cite ← Genome engineering using the CRISPR-Cas9 system — The 2013 genome-engineering protocol builds on the 2012 discovery that Cas9 is a programmable dual-RNA-guided DNA endonuclease.
- cite ← RNA-Guided Human Genome Engineering via Cas9 — RNA-guided human genome engineering applies the dual-RNA Cas9 endonuclease mechanism characterized by Jinek et al. to editing human cells.
- cite ← Multiplex Genome Engineering Using CRISPR/Cas Systems — The multiplex CRISPR paper relies on the dual-RNA-guided Cas9 endonuclease mechanism as the programmable cutting system adapted for genome engineering.