The CRISPR system has several advantages over other strategies for experimentally manipulating gene expression. First, it is relatively easy for the experimenter to design the guide RNA: it simply follows standard base pairing convention. Second, the gene to be controlled does not have to be modified; the CRISPR strategy exploits DNA sequences already present in the genome. Third, numerous genes can be controlled simultaneously. Cas9 has to be expressed only once, but many guide RNAs can be expressed in the same cell; this strategy allows the experimenter to turn on or off a whole set of genes at once.
The export of the CRISPR system from bacteria to virtually all other experimental organisms (including mice, zebrafish, worms, flies, rice, and wheat) has revolutionized the study of gene function. Like the earlier discovery of restriction enzymes, this breakthrough came from scientists studying a fascinating phenomenon in bacteria without—at first—realizing the enormous impact these discoveries would have on all aspects of biology.
Large Collections of Engineered Mutations Provide a Tool for Examining the Function of Every Gene in an Organism
Extensive collaborative efforts have produced comprehensive libraries of mutations in a variety of model organisms, including S. cerevisiae, C. elegans, Drosophila, Arabidopsis, and even the mouse. The ultimate aim in each case is to produce a collection of mutant strains in which every gene in the organism has been systematically deleted or altered in such a way that it can be conditionally disrupted. Collections of this type provide an invaluable resource for investigating gene function on a genomic scale. For example, a large collection of mutant organisms can be screened for a particular phenotype. Like the classic genetic approaches described earlier, this is one of the most powerful ways to identify the genes responsible for a particular phenotype. Unlike the classical genetic approach, however, the set of mutants is “pre-engineered,” so that there is no need to rely on chance events such as spontaneous mutations or transposon insertions. In addition, each of the individual mutations within the collection is often engineered to contain a distinct molecular “barcode”—in the form of a unique DNA sequence—designed to make identification of the altered gene rapid and routine (Figure 8–56).
Figure 8–56 Making barcoded collections of mutant organisms. A deletion construct for use in yeast contains DNA sequences (red) homologous to each end of a target gene x, a selectable marker gene (blue), and a unique “barcode” sequence approximately 20 nucleotide pairs in length (green). This DNA is introduced into yeast cells, where it readily replaces the target gene by homologous recombination. Cells that carry a successful gene replacement are identified by expression of the selectable marker gene, typically a gene that provides resistance to a drug. By using a collection of such constructs, each specific for one gene, a library of yeast mutants was constructed containing a mutant for every gene. Essential genes cannot be studied this way, as their deletion from the genome causes the cells to die. In this case, the target gene is replaced by a version of the gene that can be regulated by the experimenter (see Figure 8–52). The gene can then be turned off and the effect of this can be monitored before the cells die.