A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity
Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, Emmanuelle Charpentier
Summary
This study demonstrated that the CRISPR-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease whose target specificity is determined by a dual-RNA structure formed by a CRISPR RNA (crRNA) base-paired to a trans-activating crRNA (tracrRNA). The authors showed that Cas9 introduces site-specific double-strand breaks in target DNA, with its HNH domain cleaving the complementary strand and its RuvC-like domain cleaving the noncomplementary strand. Critically, they engineered the two guide RNAs into a single chimeric guide RNA that still directed sequence-specific cleavage, establishing the system as a programmable tool for genome editing.
Key findings
- Showed that Cas9 is guided to its DNA target by a dual-RNA structure of crRNA paired with tracrRNA, with the crRNA spacer sequence specifying the cleavage site.
- Mapped the cleavage mechanism: the HNH nuclease domain cuts the target (complementary) strand and the RuvC-like domain cuts the non-target strand, producing blunt double-strand breaks.
- Fused crRNA and tracrRNA into a single-guide RNA (sgRNA) chimera that programs Cas9 for sequence-specific DNA cleavage, demonstrating the potential for RNA-programmable genome editing.
Subjects & keywords
Cite this paper
Martin Jinek, Krzysztof Chylinski, Ines Fonfara, Michael Hauer, Jennifer A. Doudna, & Emmanuelle Charpentier (2012). A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science. https://doi.org/10.1126/science.1225829
@article{jinek2012programmable,
author = {Martin Jinek and Krzysztof Chylinski and Ines Fonfara and Michael Hauer and Jennifer A. Doudna and Emmanuelle Charpentier},
title = {A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity},
journal = {Science},
year = {2012},
doi = {10.1126/science.1225829},
url = {https://doi.org/10.1126/science.1225829}
}