Experimental Design Considerations for Efficient and Specific Gene Knock-in Using CRISPR-Cas9 for HDR Utilizing Synthetic RNAs

CRISPR-Cas9 has transformed the genome engineering world with its efficiency and ease of use. This technology has been used to create gene knockouts through the imperfect repair of Cas9-induced DNA breaks by the non-homologous end joining (NHEJ) pathway. Precise genome repair can also be achieved with the CRISPR-Cas9 system by providing a repair template, such as a single-stranded DNA oligonucleotide or double-stranded DNA plasmid, to be incorporated into the cell’s genome by the homology-directed repair (HDR) pathway. However, the efficiency of gene knock-in with the HDR pathway is much lower when compared to gene knockout with the NHEJ pathway. We set out to identify the experimental parameters that improves the efficiency of creating precise small and large insertions. We have found that one of the biggest factors to increase the likelihood of a successful, precise knock-in is optimization of transfection conditions including appropriate concentrations of transfection reagent, Cas9 nuclease, guide RNA, and repair template. Design parameters of single-stranded oligonucleotide repair templates were also systematically evaluated to define ideal length, chemical modifications, and symmetry of homology arms to knock-in a short recognition sequence. Finally, a workflow for precise tagging of a protein with a fluorescent reporter was developed including the characterization of clonal cell lines by fluorescence microscopy for expected endogenous protein localization and Sanger sequencing of the intended fluorescent reporter integration site to confirm accurate editing. Additional design and analysis considerations for precise genome engineering with HDR and CRISPR-Cas9 system will also be presented.