Creative use of the CRISPR system, discovered in bacteria as a defense against viruses, has largely solved this problem. As described in Chapter 7, the CRISPR system uses a guide RNA sequence to target (through complementary base-pairing) double-stranded DNA, which it then cleaves (see Figure 7–78). The gene coding for the key component of this system, the bacterial Cas9 protein, has been transferred into a variety of organisms, where it greatly simplifies the process of making transgenic organisms (Figure 8–55A and B). The basic strategy is as follows: Cas9 protein is expressed in ES cells along with a guide RNA designed by the experimenter to target a particular location on the genome. The Cas9 and guide RNA associate, the complex is brought to the matching sequence on the genome, and the Cas9 protein makes a double-strand break. As we saw in Chapter 5, double-strand breaks are often repaired by homologous recombination; here, the template chosen by the cell to repair the damage is often the altered gene, which is introduced to ES cells by the experimenter. In this way, the normal gene can be selectively damaged by the CRISPR system and replaced at high efficiency by the experimentally altered gene.
The CRISPR system has a variety of other uses. Its particular power lies with its ability to target Cas9 to thousands of different positions across a genome through the simple rules of complementary base-pairing. Thus, if a catalytically inactive Cas9 protein is fused to a transcription activator or repressor, it is possible, in principle, to turn any gene on or off (Figure 8–55C and D).
Figure 8–54 Transgenic mice engineered to express a mutant DNA helicase show premature aging. The helicase, encoded by the Xpd gene, is involved in both transcription and DNA repair. Compared with a wild-type mouse of the same age (A), a transgenic mouse that expresses a defective version of Xpd (B) exhibits many of the symptoms of premature aging, including osteoporosis, emaciation, early graying, infertility, and reduced life-span. The mutation in Xpd used here impairs the activity of the helicase and mimics a mutation that in humans causes trichothiodystrophy, a disorder characterized by brittle hair, skeletal abnormalities, and a very reduced life expectancy. These results indicate that an accumulation of DNA damage can contribute to the aging process in both humans and mice. (From J. de Boer et al., Science 296:1276–1279, 2002. With permission from AAAS.)
Figure 8–55 Use of CRISPR to study gene function in a wide variety of species. (A) The Cas9 protein (artificially expressed in the species of interest) binds to a guide RNA, designed by the experimenter and also expressed. The portion of RNA in light blue is needed for associations with Cas9; that in dark blue is specified by the experimenter to match a position on the genome. The only other requirement is that the adjacent genome sequence includes a short PAM (protospacer adjacent motif) that is needed for Cas9 to cleave. As described in Chapter 7, this sequence is how the CRISPR system in bacteria distinguishes its own genome from that of invading viruses. (B) When directed to make double-strand breaks, the CRISPR system greatly improves the ability to replace an endogenous gene with an experimentally altered gene since the altered gene is used to “repair” the double-strand break (C, D). By using a mutant form of Cas9 that can no longer cleave DNA, Cas9 can be used to activate a normally dormant gene (C) or turn off an actively expressed gene (D). (Adapted from P. Mali et al., Nat. Methods 10:957– 963, 2013. With permission from Macmillan Publishers Ltd.)