LEVERAGING PROGRAMMABLE INTEGRASES FOR HUMAN GENOME ENGINEERING The genetic engineering toolbox comprises a diverse array of molecular machineries for genome manipulation, and DNA insertion methods have arguably had the largest impact on biomedical research. Gene knock-ins are used in the clinic to treat genetic diseases and cancer, in industry to manufacture biologics, in agriculture to improve crops, and in research to generate models of human disease, among many other uses. These applications generally depend on either random integration mediated by viruses and transposases, or site- specific integration mediated by homologous recombination and gene editing. The former category exhibits high efficiency but little specificity, whereas the latter category is inherently precise but reliant on cellular factors and thus ineffective. Only recently has a new molecular functionality been discovered that is both fully autonomous and also highly accurate: programmable integrases directed by CRISPR RNAs. CRISPR systems have revolutionized biology over the past decade because of how easily one can program CRISPR-associated nucleases with guide RNAs to introduce DNA double-strand breaks, the precursor to DNA repair. Whereas the inability to easily redesign engineered nucleases previously stalled gene-editing technology, the discovery of RNA-guided DNA targeting eliminated this critical bottleneck. A similar bottleneck for engineered integrases is now ready for elimination. My central vision is to develop programmable, RNA-guided integrases as a powerful new platform technology for human genome engineering. Building on our recent work that deciphered sequence determinants of this technology in bacteria, as well as parallel studies that expanded the CRISPR?Cas subtypes that function robustly in mammalian cells, we will embark upon a systematic effort to build the capabilities for employing these multi-subunit integrases in eukaryotic cells. We will then develop the first tools for performing simultaneous, multiplexed DNA insertion events across thousands of distinct genomic target sites using guide RNA libraries. This approach will enable us to probe fundamental questions regarding the role of noncoding elements such as enhancers and insulators in regulating gene expression. Furthermore, we will harness orthogonal integrases to execute highly programmed translocation events and study the role of complex genome rearrangements in disease and cancer. Our studies will contribute powerful new tools to the genetic engineering toolbox and open the door to genomic manipulations that are inaccessible with any other experimental approach.
The ability to insert recombinant DNA into the genome is central to nearly all biomedical research, yet current methods for DNA integration are either non-specific or inefficient. We propose to leverage newly discovered programmable integrases for human genome engineering, in which CRISPR RNA-guided complexes direct the insertion of genetic payloads at user-defined target sites. By developing methods for high-fidelity, orthogonal, and multiplexed DNA integration, we will gain new insights into genome organization and the role of complex genome rearrangements in disease and cancer.
