Fig. A.47 Genetic engineering using the bacterial CRISPR/ Cas9 system. Genetic engineering can be targeted to a specific gene locus in cells by using two components, the bacterial Cas9 enzyme and a guide RNA (left panel). The guide RNA is a single-stranded RNA that contains two regions of sequence in tandem, the first with homology to the gene being targeted and a second recognized by the Cas9 enzyme. The guide RNA targets the enzyme to the homologous genomic region, promoting double-stranded DNA cleavage by the Cas9 endonuclease 3–4 nucleotides upstream of the protospacer adjacent motif (PAM) sequence (right panel). The PAM sequence required by the Cas9 endonuclease is the dinucleotide GG (CC on the other strand). When the double-stranded DNA break is repaired by the nonhomologous end-joining pathway, small deletions and/or point mutations are introduced into the target gene, often leading to loss of gene function. To induce a specific sequence replacement in the target gene, cells are provided with a template DNA, in addition to Cas9 and the guide RNA. This template is a double-stranded DNA sequence homologous to the target gene, but containing specific nucleotide changes. In the presence of this template, cells will repair the Cas9-mediated double-stranded cleavage using homologous recombination, rather than nonhomologous end-joining, thereby replacing the original sequence with the sequence provided in the template DNA.
by the nonhomologous end-joining DNA repair pathway, small insertions or deletions are commonly introduced, leading to a disruption of the original sequence.
This powerful technique can be used to generate homozygous gene deficiencies in cultured cells and cell lines, but importantly, it can also be used as a single-step means of generating homozygous mutant mice. For this latter purpose, RNA molecules encoding Cas9 are mixed with guide RNAs and injected into single-cell mouse zygotes, using the same technique as that used to generate transgenic mouse lines (see Fig. A.43). Due to the efficiency of the CRISPR/Cas9 system, these embryos frequently harbor mutations on both alleles of the targeted gene. Thus, after transplantation of the embryos into foster mothers, the pups born from these embryos are already homozygous for the targeted gene, without the need for lengthy mouse breeding. A refinement of this technique has been developed that allows specific nucleotide changes to be introduced into the targeted gene, rather than the random changes resulting from nonhomologous end-joining. This is accomplished by introducing a DNA oligonucleotide into the fertilized mouse zygotes along with the Cas9 and guide RNAs. The oligonucleotide contains the desired nucleotide changes flanked by sequences homologous to the targeted gene. When this oligonucleotide is present, the double-strand DNA break introduced by Cas9 is preferentially repaired by a homology-directed process that replaces the damaged DNA with the sequences from the oligonucleotide (see Fig. A.47).
A-36 Knockdown of gene expression by RNA interference (RNAi).