The current standard of Mendelian breeding strategies in the laboratory mouse means that certain genotypes comprised of multiple loci and/or two or more linked genes are impractical. This is a significant obstacle to solving a variety of problems, including production of better animal models for drug discovery and to understand complex genetic diseases and evolutionary traits. The CRISPR-Cas9 based gene drive system recently developed in insects catalyzes the conversion of heterozygous genotypes to homozygosity and biases the inheritance of a preferred allele. Despite the potential to more broadly transform basic and applied genetics, especially in rodent models, this technology has not yet been developed and implemented in a vertebrate species. The proposed objectives will leverage the innovative design of a transgenic mouse with an ?active genetic element? to establish evidence for the feasibility and efficiency of CRISPR gene drive in rodents. The CopyCat element disrupts a pigmentation gene in the mouse genome. The insert encodes a guide RNA (gRNA) designed to recognize the homologous allele in a heterozygous animal. When crossed to a mouse that expresses the Cas9 transgene, gRNA and Cas9 protein will combine to cleave DNA at the target recognition site. The resulting double strand break can then be repaired by non-homologous end joining to produce a mutation or by recombination with the homologous chromosome to convert a heterozygous CopyCat genotype to homozygosity. Gene drive efficiency will be measured as the frequency of inheritance of a copied CopyCat allele on a genetically marked target chromosome. As the first steps toward implementing CRISPR-Cas9 mediated gene drives in rodents, the proposed work will assess the frequency of inheritance of a converted gene drive allele under conditions that vary the timing of Cas9 expression.
Aim 1 of this proposal seeks to assess the efficiency of gene drive when Cas9 protein is expressed in the newly fertilized egg or in the embryo.
Aim2 will assess the efficiency of CRISPR gene drive when Cas9 protein is expressed during male and female gametogenesis when homology directed repair is naturally favored. Even if unsuccessful, these efforts will catalyze the next phase of research using the CopyCat tool to further optimize conditions that favor gene drive allele transmission in mice. If successful, this work will initiate a new era of laboratory applications for gene drives in rodents and in other vertebrates.
Although many basic and biomedical research problems could be solved by the production of laboratory rodent models with complex genotypes, traditional breeding schemes require prohibitive time, money, and numbers of animals. While a highly efficient strategy to bias the inheritance of preferred alleles has been developed in fly and mosquito, it is not yet clear whether this powerful technology can be implemented in vertebrates. This proposal leverages an innovative genetic tool to evaluate the feasibility of CRISPR-Cas9 mediated gene drive in mice in an effort to catalyze further development for a variety of applications.! !
