Figure 7.

In vitro assays to select effective Ddit3 and Sarm1 gRNAs. A, Schematic illustration of AAV vectors for U6 promoter driven gRNA expression. The C/S gRNAs include four U6 promoters that drive 2 Ddit3 and Sarm1 gRNAs individually. B, Sequences of 4 Ddit3 gRNAs and Sarm1 gRNAs that we tested in vitro. The red coded sequences are selected to make C/S gRNAs. C, Schematic illustration of mCHOP-mCherry and Sarm1-mCherry constructs and corresponding targeting regions of gRNAs. Note, 49 bp in between of C2 and C3 sgRNAs and 147 bp in between of S3 and S4 sgRNAs are potential truncations by paired sgRNAs. D, Co-transfection of SpCas9, gRNAs and reporters (Ddit3-mCherry or Sarm1-mCherry) in HEK293T cells to determine knock-down effects. Scale bar, 50 µm.