In Vitro gRNA Production Protocol

gRNAs were produced in vitro using previously published methods^{42 (link)}. Briefly, overlapping oligomers containing a T7 promoter, the desired protospacer, and gRNA scaffold were amplified using Phusion polymerase (New England Biolabs). The unpurified DNA product was then subjected to in vitro transcription using the NEB HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs), incubating at 37 °C for 16 h. The following day, RNA was treated first with DNase I followed by rSAP (recombinant Shrimp Alkaline Phosphatase; New England Biolabs), purified with the miRNeasy kit (Qiagen), concentration measured by Nanodrop, and frozen at −80 °C.