J. R. Butler1, G. R. Martens1, J. M. Ladowski1, B. W. Estes1, Z. Wang1, P. Li1, M. Tector1, A. Tector1 1Indiana University School Of Medicine,Surgery,Indianapolis, IN, USA
Introduction: Nuclease-based genome editing has rapidly sped the creation of new models of human disease. Moving forward, these techniques also hold great promise for the future of cell-based therapies for cancer, HIV and immunodeficient pathology. However, to fully realize the potential of nuclease editing tools, the efficiency and precision of their application must be optimized. The object of this study was to employ non-integrating selection and nuclease-directed homologous recombination to better control the process of genetic modification. The results have immediate application to the creation of animal models of surgical disease.
Methods: CRISPR/Cas9 directed mutagenesis with a single-guide RNA target was designed to target the GGTA1 locus of the porcine genome. A bisistronic vector expressing a single-guide RNA, Cas9 protein, and GFP was employed to increase plasmid-delivered mutational efficiency. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths to the mutational event site. These products were introduced to cells with a constant concentration of CRISPR/cas9 vector. Phenotype-specific mutational efficiency was measured by flow cytometer. Controlled homologous insertion was measured by sanger sequence and restriction enzyme digest.
Results: Bisistronic expression of a fluorescence protein on the Cas9 vector created a non-integrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5 to 82% (Figure 1A). Cotransfection with homologous DNA oligonucleotides (Figure 1B) increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic events (Figure 1C). Single-strand DNA (ssDNA) was twice as efficient as double strand DNA (dsDNA). Furthermore, these oliogos were able to effect controlled insertion with an efficiency of up to 37%. (Figure 1D-E)
Conclusion: A non-integrating selection strategy based on bisistronic fluorescence expression can increase the mutational efficiency the CRISPR/Cas9 system by greater than 2,500%. The precision of this system can be increased by the addition of a very short homologous template sequence. Together these strategies may be employed to efficiently control mutational events at unprecedented levels. This system can better utilize the potential of nuclease-mediated genomic editing.
