The accelerating acquisition of genome sequence data in diverse pioneer species has heightened the need for new genetic tools to explore gene function in pioneer organisms. Another important unmet need in control insect disease vectors and animal and plant pests is a system for dispersing effector transgenes into wild populations of these species. To help address these needs, we have developed a new method based on the CRISPR/Cas9 genome editing system referred to as the Mutagenic Chain Reaction (MCR), which results in the converts heterozygous alleles to the homozygous state in a single step. Because MCR acts efficiently in the germline as well in somatic cells, this technology is also broadly applicable to dispersing genetic elements via an extreme form of drive within a population of organisms or within cells within an organism. In this application we describe the MCR method and propose to characterize this novel genetic system in Drosophila. The MCR method is based on constructs (MCR elements) comprised of three constituents: 1) a gene encoding the bacterial Cas9 endonuclease, 2) a guide-RNA (gRNA) genes that targets Cas9 cleavage to specific genomic sites, and 3) homology arms flanking and directly abutting the gRNA directed cut sites. Cas9 and gRNA(s) carried by an MCR lead to cleavage of the genomic DNA on the other allele and the flanking homology arms result in that construct being copied into the genome via homology directed repair thereby making the MCR construct homozygous. Preliminary experiments indicate that MCR is highly efficient in both somatic and germline cells (>95%). Thus, such elements should spread exponentially through a population, initially doubling at each generation. The ability of MCR elements convert the opposing allele and to spread efficiently via the germline should be of significant value in a broad variety important applications including single step mutagenesis in pioneer organisms, accelerating genetic manipulations in all organisms, providing a potent form of genetic drive to help spread transgenes throughout populations of pest species (e.g., in mosquitoes to combat malaria), and potentially for broad delivery of genetic constructs within the body to combat diseases such as HIV, cancer, and gene therapy strategies. We also propose to develop tools to help limit the spread or eliminate MCR elements if necessary such as an Element for Reversion of the Autocatalytic Chain Reaction (ERACR). ERACR elements carry two gRNAs directing cleave of genomic sequences flanking an MCR insertion site, as well as homology arms abutting these cleavage sites, but do not carry a source of Cas9 endonuclease. ERACRs should be able to spread exponentially throughout a population carrying the targeted MCR element but should have no effect in wild-type organisms lacking a source of Cas9. In this proposal, we will characterize parameters influencing the spread of MCR elements, measure off target effects of MCR and develop opposing ERACR elements and conduct competition experiments between MCR and ERACR elements in populations.
The accelerating acquisition of genome sequence data in diverse pioneer species has heightened the need for new genetic tools to explore gene function in pioneer organisms, and another important unmet need is a system for dispersing effector transgenes into wild populations in control insect disease vectors and animal and plant pests. We have made an important step forward in providing such a technology by developing a method based on the CRISPR/Cas9 system that we refer to as the Mutagenic Chain Reaction (MCR) in which a genetic element (MCR element) is capable of spreading from one chromosome to another and by so doing can propagate itself autocatalytically via the germline with great efficiency. We propose to characterize this system genetically in the fruit fly Drosophila melanogaster and to devise various strategies to mitigate potential side effects of this system by generating elements that either remove the MCR element from a population if desired or correct any unintended mutations it may generate.
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