A problem with gene knockouts arises when the function of the gene is essential for the survival of the animal; in such cases the gene is termed a recessive lethal gene, and homozygous animals cannot be produced. To study the function of such a gene, tissue-specific or developmentally regulated gene deletion can be employed. This strategy makes use of the DNA sequences and enzymes used by bacteriophage P1 to excise itself from a host cell’s genome. Integrated bacteriophage P1 DNA is flanked by recombination signal sequences called loxP sites. A recombinase, Cre, recognizes these sites, cuts the DNA, and joins the two ends, thus excising the intervening DNA in the form of a circle. This mechanism can be adapted to allow the deletion of specific genes in a transgenic animal only in certain tissues or at certain times in development. First, loxP sites flanking a gene, or perhaps flanking just a single exon, are introduced by homologous recombination (Fig. A.46). Usually, the introduction of these sequences into flanking or intronic DNA does not disrupt the normal function of the gene. Next, mice containing such loxP mutant genes are mated with mice made transgenic for Cre recombinase that has been placed under the control of a tissue-specific or inducible promoter. When the Cre recombinase is active, either in the appropriate tissue or when induced, it excises the DNA between the inserted loxP sites, thus inactivating the gene or exon. Thus, for example, using a T-cell-specific promoter to drive expression of the Cre recombinase, a gene can be deleted only in T cells while remaining functional in all other cells of the animal. This extremely powerful genetic technique was used to demonstrate the importance of B-cell receptors in B-cell survival.
Recently, a new technology has been developed for inducing specific gene disruptions in mice; it is known as the CRISPR/Cas9 system. This technique is adapted from a bacterial system that uses an RNA-based strategy to generate double-stranded DNA breaks in the genomes of invading pathogens or plasmids, a form of bacterial immunity. The Cas9 gene encodes an endonuclease; this has been modified for use in eukaryotic cells by incorporating a nuclear localization signal into the protein-coding sequence of the enzyme. To target mutations to a particular gene, a synthetic guide RNA is produced that incorporates a short sequence (~20 nucleotides) homologous to the gene
Animals containing the loxP sites are made transgenic for the Cre protein, expressed from a tissue-specifc promoter only in certain cells, e.g., lymphocytes
When expressed, the Cre protein excises the DNA between the loxP sites, deleting the gene only in specifc cell types
Fig. A.46 The P1 bacteriophage recombination system times during development (center panel). In the cells in which the Cre can be used to eliminate genes in particular cell lineages. protein is expressed, it recognizes the loxP sequences and excises The P1 bacteriophage protein Cre excises DNA that is bounded the DNA lying between them (right panel). Thus, individual genes can by recombination signal sequences called loxP sequences. be deleted only in certain cell types or only at certain times. In this These sequences can be introduced at either end of a gene by way, genes that are essential for the normal development of a mouse homologous recombination (left panel). Animals carrying genes can be analyzed for their function in the developed animal and/or in flanked by loxP can also be made transgenic for the gene encoding specific cell types. Genes are shown as boxes, RNA as squiggles, the Cre protein, which is placed under the control of a tissue-specific and proteins as colored balls. promoter so that it is expressed only in certain cells or only at certain being targeted along with sequences that bind the Cas9 enzyme. The guide RNA recruits Cas9 to the genomic location, where the endonuclease will produce a double-stranded DNA break (Fig. A.47). When this break is repaired