Supplementary Figure 8: AsCas12a-VPR and 20-nt guides produce knockout phenotypes in vivo.

(a) Schematic of the reporter cell line employed for in vitro validation of Trp53-guide activity. A doxycycline-inducible TRE-RFP-shp53 reporter cell line was used. Dox withdrawal results in growth arrest. Trp53 loss-of-function rescues cell growth. (b) Cell growth as determined by colony formation using crystal violet (CV). 20-nt Trp53-guides were lentivirally delivered to mouse cells stably expressing AsCas12a-VPR. N = 2 independent experiments; Representative results are shown. (c) Relative mRNA expression after lentiviral delivery of 20-nt Trp53-guides to mouse cells stably expressing AsCas12a-VPR. Schematic of the approach showing a gene model not drawn to scale. Three different exon-targeting 20-nt Trp53-guides were used. Three different qPCR primer pairs located at varying loci were used to determine mRNA abundance. N = 2 independent experiments. (d) Cell growth as determined by colony formation using crystal violet (CV). Trp53-guide 3 of different lengths were lentivirally delivered to mouse cells stably expressing AsCas12a-VPR. N = 2 independent experiments; Representative results are shown. (e) Schematic depicting the in vivo experiment in which Trp53 loss-of-function results in liver tumor formation. HDTV of AsCas12a-VPR, 20-nt Trp53-guide crRNA expressing vector, c-Myc, and SB into mice. (f) Tumor-free survival. N = 3 animals per group. Rosa26-guide serves as control. (g) Livers taken 7–8 weeks post-HDTV. N = 3 mice per group. Representative images are shown. Scale bar = 1 cm. (h) Indel formation in the Trp53 locus as evidenced by T7EI assay. T7EI assay was performed with three tumor derived cell lines. Trp53 wt murine tumor cell lines were used as control (ctrl cells 1 and 2, respectively). N = 2 independent experiments; Representative results are shown. (i) Trp53 western blot. Cell lines derived from mouse tumors were treated with Doxorubicin to induce Trp53 expression. Trp53 protein levels were determined by Western blot. A Trp53-proficient mouse tumor cell line was used as control. N = 2 independent experiments; Representative results are shown.