Rapid Development of Mouse Resource Strains through the Application of Prime Editors - PROJECT SUMMARY/ABSTRACT The human genome and that of its experimental surrogate, the mouse, contain a haploid DNA content of approximately three gigabase pairs (3 X 109 bp) and an estimated 20,000 genes. Interrogating this complex landscape of genes and developing community resources around them have traditionally involved modifying the genome on a gene-by-gene and resource-by-resource basis with tremendous effort and at great cost. For example, to create an animal resource containing a knockout of each mouse gene, the International Mouse Knockout Consortium has had to target each of 20,000 unique sequences at a combined cost of close to $1 billion (USD). Contributing to the funding and labor burden is the fact that each experiment is dependent upon the technically challenging and labor-intensive microinjection of DNAs into fertilized oocytes, or embryonic stem cells into early developmental stage mouse blastocysts. Creating even a second, new genome-wide resource based on each of 20,000 genes could conceivably require repeating the costly process yet again. To move beyond the one-by-one approach for developing genome-wide resources, outlined/envisioned in this proposal, is a powerful combinatorial technology to sequentially marry functional DNA sequences (encoding, for example, reporters, recombinases, cell-ablating toxins, protein interaction domains, and nucleases) to each of thousands of unique mouse genes already marked with a uniform (lacZ-) sequence tag. The potential for a breakthrough in genome analysis studies at the heart of the proposal comes from the combination of two powerful and proven technologies — first, Prime Editing, a CRISPR technology employing Reverse Transcriptase to insert DNA sequences at points of DNA modification; and second, expression of Prime Editing machinery directly from the genome (rather than administering it exogenously). The key aspect of the proposed experiments is to exploit the exquisite specificity of Prime Editing to guide the reverse transcription of pegRNA edits/cargoes to multiple, specific, previously (e.g., lacZ-) tagged loci. Importantly, successful Prime Editing from specific pegRNAs will afford the opportunity to distribute novel functional sequences to thousands of mouse genes without any need for the costly, time-consuming microinjection of zygotes or embryonic stem cells. Full implementation of such as system has the potential to accelerate the development, increase the number, and decrease the cost of mouse genome-wide resources by orders of magnitude, and to move strain development efforts from a gene-by-gene method to a more massively parallel approach. The combinatorial nature of the technology and the adaptability of the system to incorporate the latest in new technological developments will allow a longstanding contribution to human health.
