CRISPR-Cas9 Programmable Genome Editing

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 in the type Charpentier studied, RNA transcribed from these arrays guides the Cas9 protein to matching sequences in invading viruses, which Cas9 then cuts. Emmanuelle Charpentier, working at Umea University in Sweden, discovered tracrRNA, the second RNA of the system, in Streptococcus pyogenes and published the finding in 2011. 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 central 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 adapted the system for mammalian cells within months; Feng Zhang's group at the Broad Institute reported multiplex editing in human and mouse cells in Science in early 2013. 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 Zhang's laboratory, which had filed patents on applying the system in eukaryotic cells, and the legal proceedings continued for years.

In 2018, He Jiankui announced that he had used CRISPR to edit human embryos that were subsequently born as live infants, an action widely condemned for proceeding without adequate ethical oversight. Regulatory and scientific bodies called for a moratorium on clinical germline editing pending broader international consensus. On 16 November 2023 the UK MHRA authorized exagamglogene autotemcel (Casgevy), a CRISPR-based therapy, for sickle cell disease and transfusion-dependent beta-thalassemia, and on 8 December 2023 the FDA approved it for sickle cell disease, its first approval of a treatment using this kind of genome editing. Doudna and Charpentier received the 2020 Nobel Prize in Chemistry.