Example 94
After testing the gRNAs for both on-target activity and off-target activity, the mutation correction and knock-in strategies will be tested for HDR gene editing.
For the mutation correction approach, the donor DNA template will be provided as a short single-stranded oligonucleotide, a short double-stranded oligonucleotide (PAM sequence intact/PAM sequence mutated), a long single-stranded DNA molecule (PAM sequence intact/PAM sequence mutated) or a long double-stranded DNA molecule (PAM sequence intact/PAM sequence mutated). In addition, the donor DNA template will be delivered by AAV.
For the cDNA knock-in approach, a single-stranded or double-stranded DNA having homologous arms to the 17q21 region may include more than 40 nt of the first exon (the first coding exon) of the G6PC gene, the complete CDS of the G6PC gene and 3′UTR of the G6PC gene, and at least 40 nt of the following intron. The single-stranded or double-stranded DNA having homologous arms to the 17q21 region, which includes more than 80 nt of the first exon of the G6PC gene, the complete CDS of the G6PC gene and 3′UTR of the G6PC gene, and at least 80 nt of the following intron. The single-stranded or double-stranded DNA having homologous arms to the 17q21 region may include more than 100 nt of the first exon of the G6PC gene, the complete CDS of the G6PC gene and 3′UTR of the G6PC gene, and at least 100 nt of the following intron. The single-stranded or double-stranded DNA having homologous arms to the 17q21 region may include more than 150 nt of the first exon of the G6PC gene, the complete CDS of the G6PC gene and 3′UTR of the G6PC gene, and at least 150 nt of the following intron. The single-stranded or double-stranded DNA having homologous arms to the 17q21 region may include more than 300 nt of the first exon of the G6PC gene, the complete CDS of the G6PC gene and 3′UTR of the G6PC gene, and at least 300 nt of the following intron. The single-stranded or double-stranded DNA having homologous arms to the 17q21 region may include more than 400 nt of the first exon of the G6PC gene, the complete CDS of the G6PC gene and 3′UTR of the G6PC gene, and at least 400 nt of the following the first intron. Alternatively, the DNA template will be delivered by AAV.
Next, the efficiency of HDR mediated correction of the common mutation of G6PC R83 and knock-in of cDNA into the 1st exon will be assessed.
