2.4 In vitro transcription for sgRNA synthesis
pIVEX_sgRNA vectors were digested with Dra I (NEB) to facilitate
run-off sgRNA synthesis. After digestion, the enzyme was inactivated by
incubation at 65 °C for 20 minutes, and the DNA was purified using the
MSB Spin PCRapace kit (Invitek Molecular GmbH, Berlin) according to the
manufacturer´s instructions. In vitro transcription was performed
with 1 µg of purified DNA template using the HiScribe T7 High-Yield RNA
Synthesis Kit (NEB). Reactions were incubated for four hours at 37 °C,
and the RNA samples were stored at -20 °C until use.