In vitro transcription of guide RNAs and 5'-triphosphate removal
Mark Dewitt, Julia Wong, Beeke Wienert, Moritz F Schlapansky, Eric Aird
Abstract
sgRNA template assembly, in vitro T7 transcription, and sgRNA column cleanup to remove 5'-triphosphate groups
Before start
Steps
sgRNAs need to be purfied before dephosphorylation and transfection. There are different methods one could purify their sgRNAs. We therefore tested different purification kits and found that the Qiagen miRNeasy Tissue/Cells Advanced Mini Kit gives the most consistent and highest yields. To purify our sgRNAs with this kit, we follow the manufacturer's instructions with the following modifications:
Adjust sgRNA sample to a volume of 100 µl with RNase-free water.
Add 350 µl RLT Buffer to the sample and mix well by pipetting
Add 450 µl Isopropanol and mix well by pipetting.
Transfer sample (~900 µl) to an RNeasy mini spin column; spin for 15 sec at 10.000 g.
Discard the flow-through.
Add 700 µl RWT Buffer; Spin for 15 sec at 10.000 g.
Discard the flow-through.
Add 500 µl RPE Buffer; Spin for 2 min at 10.000 g.
Move spin column to a new collection tube and spin for 1 min at 10.000 g to dry the membrane completely.
Move spin column to an RNAse-free 1.5 ml microfuge tube
Add 33 µl DEPC-treated H2O; spin 1 min
Optional: Repeat the elution to collect any remaining RNA on the column and increase RNA concentration.
