FIGURE 6-43 Single-nucleotide mutations can be introduced into the genome using an engineered CRISPR-Cas9 system. (a) The genome of a target cell can be modified by expression of the double-stranded DNA endonuclease Cas9 and a guide RNA. Expression of these components can be achieved by transfection with plasmids carrying genes for Cas9 and the guide RNA or by direct injection of Cas9 mRNA and guide RNA. The guide RNA is composed of two parts: a sequence that folds into a hairpin scaffold structure that binds to Cas9, and a sequence of approximately 20 nt corresponding to the targeted site in the genome. Expression of these components can be achieved by transfection with plasmids carrying genes for Cas9 and the guide RNA or by direct injection of Cas9 mRNA and guide RNA. (b) A complex of guide RNA bound to Cas9 is targeted to the genome by base pairing of the guide RNA with the complementary genomic DNA sequence. This structure allows the two distinct nuclease active sites of Cas9 to cleave both strands of the target DNA adjacent to the heteroduplex formed with the guide RNA. (c) By this mechanism, the expression of both Cas9 and a bipartite guide RNA designed to target a specific gene sequence leads to a double-strand cleavage of the target gene. (d) Cleaved DNA can be repaired via a nonhomologous end joining (NHEJ) process, which usually removes a small number of bases at the cleavage site. If the cleavage occurs in a coding sequence, NHEJ will usually inactivate gene function by producing a frameshift mutation. If a ∼100-nt single-stranded DNA segment that spans the sequences flanking the cleavage site is injected along with Cas9 mRNA and the guide RNA, the cleaved DNA can be repaired by homologous recombination (homology-directed repair, HDR). By this mechanism, single base changes can be introduced into the repaired genomic DNA.

[Part (b) data from C. Anders et al., 2014, Nature 513:569-573, PDB ID 4un3.]