CRISPR, short for clustered regularly interspaced short palindromic repeats, is a genome engineering method that enables rapid and precise modification of nearly any genome. In nature, CRISPR systems provides adaptive immunity to bacteria and archea. In the simplest CRISPR systems, RNAs containing sequence complementary to viral or plasmid DNA interact with the DNA nuclease Cas9 to direct sequence-specific cleavage of invading DNA.
Recognizing the potential of harnessing this system for genome engineering in other organisms, in 2012 Jinek and colleagues identified a minimal two-component system capable of inducing the site-specific cleavage of DNA: Cas9 and a chimeric guide RNA (gRNA) from S. pyogenes. Straightforward and readily implemented, CRISPR-based genome engineering has been rapidly adopted by research community. By 2013, we and many others had demonstrated that CRISPR could be used to edit the genome of mammalian cells, zebrafish, Drosophila and more.
Inducing double-strand DNA breaks with CRISPR forces the cell to initiate DNA repair, opening a window of opportunity for modifying the original sequence during the repair process. Cells employ two major pathways to repair double-strand DNA breaks and both can be co-opted for genome editing. Non-homologous end joining (NHEJ) and related pathways repair broken DNA ends by ligation, an imprecise process that can result in insertions and deletions (called indels) at the breakpoint. Targeting the NHEJ pathway is a straightforward way to generate loss-of-function alleles by targeting breaks to critical genomic sequences and selecting for disruptive repair events. In contrast, homology-directed repair (HDR) uses homologous DNA as a template for DNA synthesis to bridge the gap across a double-strand break. By providing a donor repair template, researchers can appropriate HDR to introduce precise sequence modifications or new DNA sequences.