Optimized gRNA Transcription Protocol

Templates for gRNA transcription with T7 polymerase were generated by two-oligo PCR, using one oligo containing the T7 promoter and gene-specific sequence with overlap to a second reverse complement oligo containing the constant region of the gRNA backbone. An optimized gRNA constant region was used24 (link), except in Supplementary Fig. 13 where two constant regions are compared for one gRNA. As in previous work8 (link)25 (link), the bases at the first two positions in the gRNA were always substituted for guanine to accommodate preferences of T7 RNA polymerase. The product was purified with the E.Z.D.A. Cycle-Pure kit (Omega Bio-tek, Norcross, GA). All gRNAs were transcribed with the Megascript T7 kit (Thermo Fisher Scientific, Waltham, MA), using half-size (10 μl) reactions and DNase treated. Purification of the gRNA was either done with ammonium acetate/ethanol precipitation or with column purification, using RNA Clean & Concentrator columns (Zymo Research, Irvine, CA), if precise quantification of gRNA concentration was required (Fig. 2). For column-purified gRNAs, RNA concentration was determined using a Nanodrop spectrophotometer and all gRNAs were visualized with agarose gel electrophoresis. The random RNA control was concentration matched to gRNAs and similarly sized, as well as equivalently column purified.