Identifying and Designing New Strategies for Enhanced Genome Engineering in Ag Related Systems

CRISPR RNA-guided adaptive immune systems that protect bacteria and archaea from infection by viruses are now being routinely repurposed for genome engineering in a wide variety of cell types and multicellular organisms1-3. This new gene knockout system is revolutionizing molecular biology. However, the next major advance will be to create methods that allow efficient and precise repair of defective genes or the site-specific introduction of novel DNA containing genes with new functions. This is an active area of investigation with new innovations that include base-editing technology 119, and Cas9-fusions designed to deliver a DNA donor to the site of repair 120-123.Here we propose two complementary gain-of-function screens independently designed to identify novel pathways that enhance Homology Directed Repair (HDR) of double-stranded DNA breaks (Figure 2). This approach is distinct from previously published efforts that focus on engineering new versions of Cas9, and instead focuses on identifying genes that limit HDR. Results from this screen will provide fundamental new insight into DNA repair mechanisms in eukaryotic cells and provide a foundation for designing the next generation of tools for enhanced manipulation of complex genomes.The Specific Objectives (SO) of this proposal are to:Specific Objective 1 - Establish a human genome wide knockout library for phenotypic screening. An overview of our gain-of-function screens are shown in Figure 2. The human Genome-scale CRISPR KnockOut (GeCKO) library was purchased from Addgene. The library has been amplified by a solid agar/colony method as previously described124-126. To produce lentivirus, HEK293T cells we have transfected with 20μg of the lentiCRISPR plasmid library, 10 μg of pVSVg (env protein), and 15 μg of psPAX2 (gag, pol, rev, and tat genes) (Addgene) using Lipofectamine 3000. The lentiviral GeCKO library was concentrated and HEK293 cells were infected at an MOI of 0.2. Puromycin was used to select for transduced cells and total DNA was isolated for amplification of the sgRNA library and the library has been sequenced using an illumina platform (WSU-Spokane Genomics Core).Specific Objective 2 - Establish gain-of-function screens using the Cas9/sgRNA system to identify genes that limit Homology Directed Repair (HDR). We have engineered two cell lines to perform the gain-of-function screens designed to identify novel pathways that enhance Homology Directed Repair (HDR). One screen relies on the HDR-mediated repair of a defective fluorescent protein (i.e., GFP) and the other is a survival-based screen that relies on repair of a defective drug resistance marker (i.e., neomycin resistance gene). The GFP repair reporter integrated into HEK293 cells that stably express Cas9, and the defective neomycin resistance gene is in a haploid cell line (eHAP) that stably expresses Cas9. These cells have been transduced with the lentiviral library, selected with puromycin, and then transfected with a plasmid containing an sgRNA designed to target the defective reported and a DNA donor designed to restore reporter function (Figure 2). Cells that repair reporter function were recovered after challenge with G418 (neomycin resistant) or by FACS (GFP). Total DNA was isolated, integrated sgRNA sequences were PCR amplified from the genome, the products were deep-sequenced using an illumina platform and the data were analyzed using MaGeCK 128 (Figure 3).Specific Objective 3 - Systematically edit individual genes identified in the genome-wide screen to demonstrate their role in enhancing HDR. The annotated deep sequencing data from the high-throughput screen is designed to reveal genes that enhance HDR. To confirm the importance of these genes in HDR, we will design 3 sgRNAs that target distinct regions in each of the most abundant genes identified from the high-throughput screen. These sgRNA expression vectors will be used to transfect egfp* defective HEK293 cells expressing Cas9. Knockout lines will be cloned and rates of homologous recombination will be measured using our gain-of-function assays by restoring the function of a defective egfp* gene. These rates will be compared to the rates of HDR in egfp* HEK293 cells that have not been transfected with the sgRNAs targeting specific genes implicated in enhanced HDR.