James R Butler1, Rafael M N Santos1, Gregory R Martens2, Joseph M Ladowski3, Zheng-Yu Wang3, Ping Li1, Matthew Tector3, A Joseph Tector4. 1. Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana. 2. Department of Surgery, Indiana University School of Medicine, Indianapolis, Indiana; Department of Surgery, The University of Alabama at Birmingham, Birmingham, Alabama. 3. Department of Surgery, The University of Alabama at Birmingham, Birmingham, Alabama. 4. Department of Surgery, The University of Alabama at Birmingham, Birmingham, Alabama. Electronic address: jtector@uab.edu.
Abstract
BACKGROUND: Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or 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 use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. METHODS: Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5' to a nuclease cut 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, restriction enzyme digest and flow cytometry. RESULTS: Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic events. Single-strand DNA was twice as efficient as double-strand DNA. Furthermore, nuclease-mediated insertion by homology-directed repair successfully drove locus-specific transgene expression in the porcine genome. CONCLUSIONS: A nonintegrating selection strategy based on fluorescence expression can increase the mutational efficiency of the CRISPR/Cas9 system. The precision of this system can be increased by the addition of a very short homologous template sequence and can serve as a method for locus-specific transgene delivery. Together these strategies may be used to efficiently control mutational events. This system may be used to better use the potential of nuclease-mediated genomic editing.
BACKGROUND: Nuclease-based genome editing has rapidly sped the creation of new models of human disease. These techniques also hold great promise for the future of clinical xenotransplantation and cell-based therapies for cancer or 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 use nonintegrating selection and nuclease-directed homologous recombination to efficiently control the genetic modification of the porcine genome. METHODS: Clustered randomly integrating spaced palindromic repeats and associated Cas9 protein (CRISPR/Cas9)-directed mutagenesis with a single-guide RNA target was designed to target the alpha-1,3-galactosyltransferase locus (GGTA1) of the porcine genome. A vector expressing a single-guide RNA, Cas9 protein, and green fluorescent protein was used to increase plasmid-delivered mutational efficiency when coupled with fluorescence sorting. Single and double-strand DNA oligonucleotides with a restriction site replacing the start codon were created with variable homology lengths surrounding the mutational event site. Finally, a transgene construct was flanked with 50 base pairs of homology directed immediately 5' to a nuclease cut 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, restriction enzyme digest and flow cytometry. RESULTS: Expression of a fluorescence protein on the Cas9 vector functioned as a nonintegrating selection marker. Selection by this marker increased phenotype-silencing mutation rates from 3.5% to 82% (P = 0.0002). Insertion or deletion mutation increased from 11% to 96% (P = 0.0007). Co-transfection with homologous DNA oligonucleotides increased the aggregate phenotype-silencing mutation rates up to 22% and increased biallelic events. Single-strand DNA was twice as efficient as double-strand DNA. Furthermore, nuclease-mediated insertion by homology-directed repair successfully drove locus-specific transgene expression in the porcine genome. CONCLUSIONS: A nonintegrating selection strategy based on fluorescence expression can increase the mutational efficiency of the CRISPR/Cas9 system. The precision of this system can be increased by the addition of a very short homologous template sequence and can serve as a method for locus-specific transgene delivery. Together these strategies may be used to efficiently control mutational events. This system may be used to better use the potential of nuclease-mediated genomic editing.