Exciting new developments that permit exquisitely specific genome editing stand to usher in an era of molecular revolution. These advances come from a wholly unexpected source: the discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas (or CRISPR-associated genes). These are linked genetic elements that endow prokaryotes with a form of acquired immunity to phages and plasmids. Bacteria use this system to sample the DNA of infecting agents, incorporating it into the host genome as CRISPRs. CRISPRs are transcribed and processed into an RNA sequence that binds and directs the nuclease Cas9 to a sequences (e.g., a phage), leading to its cleavage and the destruction of the phage. Gene editing repurposes this process by using artificial guide RNAs (gRNAs) that bind Cas9 and are complementary to a DNA sequence of interest. Once directed to the target sequence by the gRNA, Cas9 induces double-strand DNA breaks.
Repair of the resulting highly specific cleavage sites can lead to somewhat random disruptive mutations in the targeted sequences (through nonhomologous end joining [NHEJ]), or the precise introduction of new sequences of interest (by homologous recombination). Both the gRNAs and the Cas9 enzyme can be delivered to cells with a single easy-to-build plasmid (
Fig. 1.5 ). However, the real beauty
DNA with random mutation DNA with specific mutation
Fig. 1.5 Gene editing with clustered regularly interspersed short palindromic repeats (CRISPRs)/Cas9. In bacteria, DNA sequences consisting of CRISPRs are transcribed into guide RNAs (gRNAs) with a constant region and a variable sequence of about 20 bases. The constant regions of gRNAs bind to Cas9, permitting the variable regions to form heteroduplexes with homologous host cell DNA sequences. The Cas9 nuclease then cleaves the bound DNA, producing a double-stranded DNA break. To perform gene editing, gRNAs are designed with variable regions that are homologous to a target DNA sequence of interest. Coexpression of the gRNA and Cas9 in cells leads to efficient cleavage of the target sequence. In the absence of homologous DNA, the broken DNA is repaired by non homologous end joining (NHEJ), an error-prone method that often introduces disruptive insertions or deletions (indels). By contrast, in the presence of a homologous “donor” DNA spanning the region targeted by CRISPR/Cas9, cells instead may use homologous DNA recombination (HDR) to repair the DNA break. HDR is less efficient than NHEJ, but has the capacity to introduce precise changes in DNA sequence. Potential applications of CRISPR/Cas9 coupled with HDR include the repair of inherited genetic defects and the creation of pathogenic mutations.