Gene editing by CRISPR–Cas

One of the most revolutionary developments in biology has its origins in the RNA-based defence system of bacteria, which encodes clustered regularly interspaced short palindromic repeats (CRISPR) along with CRISPR-associated (Cas) proteins. CRISPR–Cas has been adapted to function as a programmable genome-engineering tool that has enabled easy targeting and manipulation of precise genomic sequences in bacteria, plants, fungi and mammals, including humans.

Prior work with programmable genome-engineering tools focused on the use of various nucleases for gene targeting. Though precise, the reliance on protein-based recognition of a target DNA sequence meant that each new target required redesigning the tool, which is often a laborious task. CRISPR–Cas has the advantage of being precise and highly adaptable. This precision is derived from base-pairing between the complementary CRISPR–Cas guiding RNA and target DNA, which creates a straightforward and easy‑to‑adapt targeting system.

Foundational work was performed in 2012 by groups studying the function of CRISPR and the associated protein Cas9 in bacteria. It was known that these CRISPR–Cas systems function as a bacterial immune system against viruses. Jinek et al., and complementary work from Gasiunas et al., demonstrated that two small RNAs — the CRISPR RNA (crRNA) in a complex with a trans-activating CRISPR RNA (tracrRNA) — function together in targeting the nuclease Cas9 to specific DNA sequences, where it can generate a double-stranded DNA break (DSB). Jinek et al. had the insight that the two RNAs could be joined together to form a single guide RNA (sgRNA). The groups speculated that RNA could also be used to guide Cas9 to specific genomic sequences and thus enable gene editing and genome engineering.

The concept was demonstrated in a set of papers published in early 2013, when Jinek et al., Mali et al. and Cong et al. adapted the bacterial CRISPR–Cas system to function in mouse and human cells. Using sgRNAs and optimising the system for expression in a mammalian context, they demonstrated that Cas9 could be guided to specific target sequences in the larger and more complex mammalian genomes where, as in bacteria, it cleaved the DNA.

Mammalian cells attempt to rapidly repair these DSBs and this process can be co‑opted for tailored genome engineering. The non-homologous end-joining DSB repair pathway attempts to stitch the DNA ends back together, often resulting in the introduction of deleterious mutations in genes. Furthermore, if a DNA template is available the cell will attempt to copy genetic information from it during repair, thereby inserting new genetic information into the genome, as demonstrated by Mali et al. and Cong et al.

From its origins as a bacterial immune system, CRISPR–Cas has been developed into an all-purpose tool for tailored engineering of genomes in a range of species. In the span of less than a decade, CRISPR–Cas has opened up new avenues in our understanding of how cells repair DNA damage, in our ability to engineer cells and in the possibility of developing new, RNA-dependent therapies for previously intractable genetic diseases.