Genetics & Molecular · 2012
CRISPR-Cas9 Programmable Genome Editing
Clustered Regularly Interspaced Short Palindromic Repeats with CRISPR-associated protein 9
Precise genome editing existed before CRISPR, but it was slow and expensive enough to limit its use to specialized laboratories with dedicated resources. Zinc-finger nucleases and transcription activator-like effector nucleases could cut specified DNA sequences, but designing each new construct required weeks of protein engineering and was not always reliable. The barrier to entry kept the technology largely in the hands of large biotechnology companies and well-funded academic groups.
The biochemical foundation for CRISPR came from an unexpected source: bacterial adaptive immunity. Bacteria incorporate short fragments of viral DNA into their own genome in arrays called CRISPRs, and they use RNA transcribed from these arrays, guided by the Cas9 protein, to recognize and cut matching sequences in invading viruses. Emmanuelle Charpentier, working at Umea University in Sweden, characterized the two-RNA mechanism of the system in Streptococcus pyogenes. Her collaboration with Jennifer Doudna at the University of California, Berkeley combined structural and biochemical approaches to engineer a simplified single guide RNA that could direct Cas9 to any target sequence.
A paper published in Science in August 2012, with Martin Jinek as first author, demonstrated that the two-component system could cut purified DNA at specified sites in vitro. The key finding was reprogrammability: changing the 20-nucleotide targeting sequence of the guide RNA was sufficient to redirect Cas9 to any new target. Double-strand cleavage could then be exploited either to disrupt a gene through non-homologous end joining or to insert a precise edit using a supplied repair template.
Laboratories around the world adapted the system for mammalian cells within months of publication, and the speed of adoption was faster than for any previous editing technology. CAR-T cell trials using CRISPR-edited lymphocytes opened within a few years. The technology also generated a contentious patent dispute between the Doudna-Charpentier group and Feng Zhang's laboratory at the Broad Institute, which had filed patents on applying the system in eukaryotic cells. The legal proceedings continued for years and highlighted the commercial stakes of the underlying science.
In 2018, He Jiankui announced that he had used CRISPR to edit human embryos that were subsequently born as live infants, an action condemned by the scientific community for proceeding without adequate ethical oversight. Regulatory and scientific bodies called for a moratorium on clinical germline editing pending broader international consensus. In late 2023, the FDA and UK MHRA approved exagamglogene autotemcel, a CRISPR-based therapy for sickle cell disease and transfusion-dependent beta-thalassemia, the first licensed medicine using the technology. Doudna and Charpentier received the 2020 Nobel Prize in Chemistry.
Key People
- Jennifer Doudna — Co-developer of CRISPR-Cas9 as a programmable editing tool; 2020 Nobel laureate.
- Emmanuelle Charpentier — Co-developer; characterized the two-RNA guide mechanism; 2020 Nobel laureate.
- Martin Jinek — First author of the 2012 Science paper; demonstrated in vitro DNA cleavage.
- Feng Zhang — Adapted CRISPR-Cas9 for mammalian cells at the Broad Institute.
Science. 2012;337(6096):816-821.
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