CasRx17, specifically NLS-RfxCas13d-NLS (Addgene, 109049), was used in combination with target-specific gRNAs for KD of targeted transcripts. A deep-learning-based tool66 was used to design gRNA spacer sequences targeting the genes of interest. These spacer sequences were filtered to exclude homopolymers (more than four As, more than three Cs, Gs or Ts). Spacer sequences with potential off-target interactions were identified using BLAST67 and excluded. For each target gene, three separate spacer sequences were selected and combined into a tandem pre-gRNA array, with each spacer sequence preceded by the CasRx direct repeat (DR36) sequence66: CAAGTAAACCCCTACCAACTGGTCGGGGTTTGAAAC. Tandem pre-gRNA arrays were processed by CasRx into individual gRNAs for KD17. See Supplementary Table 1 for full sequences. Molecular cloning To create constructs for the initial NPC KD experiments, we used the lentiviral pSicoR plasmid68 (Addgene, 11579) as the backbone. The mouse U6 promoter was replaced with the human U6 promoter, which was used to drive expression of the CasRx gRNA arrays. These constructs were used in combination with a CasRx knock-in cell line (described below). To obtain enough NPCs for CasRx KD validation by auto-western blot (described below), an all-in-one construct was created that contained CasRx and EGFP expressed from the CAG promoter element and...

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