INCREASING GENE EDITING EFFICIENCIES IN EUKARYOTIC CELL LINES BY SELECTION OF APPROPRIATE CRISPR-CAS9 REAGENTS

Genetic engineering of living cells is critical for understanding gene function in normal and diseased states. The CRISPR-Cas9 system is widely utilized because of its ease-ofuse compared to other gene editing methods. This system requires a complex of Cas9 protein with tracrRNA and a gene-targeting crRNA to introduce double-strand DNA breaks at specific locations in the genome to disrupt protein translation and knockout gene function. To achieve high gene editing efficiencies, it is essential to choose the best CRISPR-Cas9 reagents for delivery and expression in the cells of interest. Depending on the transfectablity of specific cells, CRISPR components can be delivered using plasmid transfection or lentiviral transduction. Plasmid-expressed Cas9 can be co-transfected with synthetic crRNA and tracrRNA for efficient gene editing in cells amenable to lipid delivery. Cas9 that is packaged into lentiviral particles can be transduced into cells that are refractory to transfection. Lentiviral Cas9 can also be used to generate stable cell lines, which can then be transfected or transduced by synthetic or lentiviral-based CRISPR RNA components; this is particularly useful for screening applications. Importantly, expressing humanized Cas9 from different promoters (e.g., human and mouse CMV and EF1α, PGK, CAG) in different cell types results in varying levels of Cas9 expression and consequently, varying efficiencies of gene editing. In addition, cells transfected or transduced with gene editing reagents can be enriched by antibiotic selection or FACS using reagents in which the Cas9 gene is co-expressed with either an antibiotic resistance marker or a fluorescent protein reporter. This enrichment facilitates the isolation of clonal cells containing the desired mutation. Presented here are data demonstrating improvement of gene editing efficiencies in cells of interest by using the most effective delivery and selection approaches with the optimal CRISPRCas9 reagents. Lentiviral Cas9 combined with synthetic crRNA:tracrRNA Enrichment of cell populations for increased gene editing events