Fig. 2: Characterization of the GA-inducible split Cas13d (N507/508 C).

A The schematic shows the detailed design and reconstituted state of N507-GAI-NLS/GID-508C-NLS in the presence of GA, gRNA, and target transcripts. This design achieved 77% mCh-specific knockdown in transient transfection conditions. B GA-induced endogenous gene knockdown N507-GAI-NLS/GID-508C-NLS is reversible. HEK293FT cells were integrated with a PiggyBac transposon expression cassette containing the inducible Cas13. The sorted clones that highly express the inducible system were transduced with non-target (NT) gRNA or B2M targeting gRNA lentivirus. B2M knockdown was measured as HLA surface expression. The percent of HLA-positive cells were gated using an unstained control on the day of flow cytometry. The entire population lost HLA expression in 2–3 days of GA induction, which was fully recovered by day 4 after the withdrawal of the inducer. A second GA induction robustly led to the loss of HLA expression. Sample size n = 3 biological replicates. C A schematic showing the gRNA array processing by split Cas13. Guide RNA arrays were expressed as a single transcript containing spacers joined together with RfxCas13d-specific direct repeat regions. Under GA-induction, split Cas13d becomes reconstituted. They are then actively coupled with processing gRNA arrays to mature gRNAs for targeted knockdown of specific transcripts. Sample size n = 3 biological replicates. D With a gRNA array targeting B4GALNT, B2M, and CD46, the GA-inducible split Cas13d knocked down all 3 target expressions in a GA-responsive manner with efficiency comparable to that achieved using the single mature gRNA. In addition, the GA-induced single target knockdown was specific as the expression of untargeted transcripts is intact; P-value from left to right: p = 0.00157, p = 0.00226, p = 0.00507, p = 0.00467. E GA induced over 80% mCh knockdown in a neuron cell line, Neuro2A, in transient transfection conditions. Sample size n = 3 biological replicates. F GA induced 67% mCh knockdown in an immune cell line, Jurkat T cell, in transient transfection conditions. Sample size n = 6 biological replicates. G Primary cells, PBMCs, were sequentially transduced with 2 lentiviruses, each carrying the N-terminal construct or the C-terminal construct with a gRNA expression cassette. CD46 surface expression was inducibly knocked down in 33% of transduced PBMC. Sample size n = 3 biological replicates. P-values were calculated as a 2-tailed t-test. Data are presented as mean values +/− SEM. Error bars indicate the SEM for at least three biological replicates (n ≥ 3). Source data are provided as a Source Data file.