Table 5. In vitro transcription of RGR to produce gRNA molecules .

Total	20 µl
5x transcription buffer	4 µl
100 mM DTT	2 µl
~20 U/µl recombinant RNasin RNase inhibitor	0.5 µl
2.5 mM each rNTPs	4 µl
Template DNA (PCR fragment) (10-50 ng/µl [final])	x µl
T7, SP6 or T3 RNA polymerase	1 µl
H2O	(8.5 - x) µl

Note: For higher yield, add 1 U inorganic pyrophosphatase into the reaction and incubate overnight.