The pX330 plasmid for Cas9 and guide RNA expression was acquired from Addgene [13 (link)]. The pMAX plasmid for GFP expression was acquired from Lonza. Guide DNA sequences were synthesized by Integrated DNA Technologies (Newark, NJ) and phosphorylated using PNK (New England Biolabs #M0201L). They were then ligated into a BbsI (New England Biolabs #R0539L) digested pX330 plasmid using T4 DNA Ligase (New England Biolabs #M0202L). Guide integration was confirmed by sequencing.
Plasmids containing guide RNA sequences were chemically transformed into DH5α E. coli cells (ThermoFisher #18265017) and grown in LB broth (ThermoFisher #12780052) supplemented with ampicillin (ThermoFisher #) for a final concentration of 100 μg/ml. Large cultures were harvested and plasmid DNA was extracted using the endotoxin-free plasmid Maxi kit (QIAGEN #12362).
A donor DNA repair template containing a proof-of-principle, an exogenous fluorescent gene(green fluorescent protein, GFP) was constructed to be homologous to the RhD locus. Using a plasmid vector from the pCR™-Blunt II-TOPO® kit (Invitrogen, Carlsbad CA), the GFP gene and a bovine growth hormone polyA sequence were ligated respectively into the vector with arms of homology of approximately 500bp of the human RhD target site. This plasmid was transformed and harvested in the same manner as the guide RNAs.
Plasmids containing guide RNA sequences were chemically transformed into DH5α E. coli cells (ThermoFisher #18265017) and grown in LB broth (ThermoFisher #12780052) supplemented with ampicillin (ThermoFisher #) for a final concentration of 100 μg/ml. Large cultures were harvested and plasmid DNA was extracted using the endotoxin-free plasmid Maxi kit (QIAGEN #12362).
A donor DNA repair template containing a proof-of-principle, an exogenous fluorescent gene(green fluorescent protein, GFP) was constructed to be homologous to the RhD locus. Using a plasmid vector from the pCR™-Blunt II-TOPO® kit (Invitrogen, Carlsbad CA), the GFP gene and a bovine growth hormone polyA sequence were ligated respectively into the vector with arms of homology of approximately 500bp of the human RhD target site. This plasmid was transformed and harvested in the same manner as the guide RNAs.