In vitro sgRNA transcription and purification
PCR products containing sgRNA sequence under the control of a T7
promoter were synthesized using Q5 polymerase (NEB). These PCR products,
which contained the T7 promoter, were then utilized for an in vitro
transcription (IVT) reaction utilizing the HiScribe Quick T7 High Yield
RNA synthesis kit (NEB), following the manufacturer’s protocol.
Following an overnight incubation, the IVT reaction was treated with
DNAse I (NEB) for 15 min at 37°C, followed by purification using
isopropanol. The resulting pellets were air-dried and then resuspended
in RNase-free water before being stored at -80°C