Supplementary Figure 2: AsCas12a-VPR-mediated reporter activation is dictated by guide length.

(a) Schematic of the TRE-RFP reporter system. Mouse cells stably expressing TRE-driven RFP and the reverse Tetracycline transactivator (rtTA3) (Tet-on system) are used. Each TRE-guide has seven potential target sites within the TRE promoter. (b) Schematic of the reporter cell line employed. A doxycycline-inducible TRE-RFP-shp53 reporter cell line was used. Dox withdrawal results in growth arrest and loss of RFP expression. AsCas12a-VPR-mediated transcriptional activation using TRE-targeting guides induces RFP expression and rescues cell gowth in off Dox conditions. Dox = doxycycline. (c) PCR of the TRE promoter using genomic DNA obtained 10 days post-lentiviral transduction. A doxycycline-inducible TRE-RFP reporter was used to assess transcriptional activation in mouse cells stably expressing AsCas12a-VPR (right) or AsCas12a (left) and crRNAs. TRE-guide length was varied from 20-nt to 12-nt. crRNAs were lentivirally-transduced. ctrl = Rosa26-guide. N = 2 independent experiments; Representative images are shown. (d) Flow cytometry-based RFP quantification and in reporter cell lines stably expressing AsCas12a-VPR (left) or AsCas12a (right) transduced with crRNAs expressing different guide lengths (20-nt to 12-nt) targeting the TRE promoter in off Dox and on Dox conditions. Mean ± s.d.; N = 3 independent experiments. (e) Cell growth as determined by colony formation using crystal violet (CV). Reporter cell lines stably expressing AsCas12a-VPR (left) or AsCas12a (right) were transduced with crRNAs expressing different guide lengths (20-nt to 12-nt) targeting the TRE promoter and grown under off DOX conditions for 14d. ctrl = Rosa26-guide. N = 2 independent experiments. Representative images are shown.