Analysis of active genetic elements in germline and somatic cell lineages - Project Summary: Ten years ago, my laboratory and collaborators pioneered the field of active genetics , in which CRISPR-based genetic systems bypass the fundamental rules of traditional genetics. Briefly, a transgenic cassette expresses the Cas9 endonuclease and a guide RNA (gRNA) that directs DNA cleavage precisely at the site of its genomic insertion. In the germline of heterozygous carriers, the Cas9/gRNA complex cuts the opposing homolog chromosome, and the cassette is copied onto the homologous site through efficient homology directed repair (HDR). Spreading through populations in a super-Mendelian fashion, these “selfish” genetic entities, often referred to as gene-drives, offer a variety of potential applications for combatting insect vector borne diseases, crop pests, and insecticide resistance. Gene-drives can be used either to reduce insect numbers (suppression drives) or to modify insect populations to render them harmless (modification drives). Gene-drives can also be used to bias inheritance of a beneficial or favored allelic variant. For example, such allelic-drives can be used to revert a common insecticide resistant allele of the voltage gated sodium ion channel back to wild-type sensitivity in freely mating populations. These drive systems offer a new avenue to combat vector-borne diseases such as malaria, or potentially to curtail agricultural damage and enhance food security. Active genetic systems also can function in somatic cells of the body to copy gene cassettes or specific favored allelic variants from one chromosome to its homolog. Such interhomolog somatic copying could potentially restore function of disease- causing heterozygous mutant alleles using sequences copied from the intact chromosome. My proposed research program will focus on developing new active genetic systems functioning in both germline and somatic cell contexts. For example, we will develop and analyze the dynamics of transiently acting self-eliminating allelic-drive systems to reverse insecticide resistance while leaving no transgenic imprint behind. Studies will also include a new linked semi-drive system in which a driving gene cassette is inserted into an intron close to the allelic editing site, which results in the gene cassette being co-driven at intermediate levels along with the preferred allele. An important research area will be to understand mechanisms contributing to the efficiency of gene-cassette copying including a requirement for two-sided homology flanking drive elements. Another important arena of investigation will be to explore factors determining the respective efficiencies of Cas9- and scarless Nickase-induced copying in somatic and germline lineages. We will also develop models in Drosophila and human cells for correcting disease alleles by interhomolog repair, and tools to image DNA repair components in situ. Such inquiries will address how distinct DNA repair programs elicited by Cas9 versus Nickases result in alternative interhomolog conversion outcomes in differing developmental and cellular contexts. These combined studies should provide mechanistic insights active genetic processes and offer impactful applications for next-generation gene-drives and scarless genetic therapies.
