Fig. 2

Inducible sequential mutagenesis in murine cells through Cpf1-Flip. a Schematic for PCR-based detection of Cre-mediated inversion of the crRNA FlipArray (targeting Nf1 and Pten). b PCR-based detection of non-inverted and inverted FlipArrays at D0 (n = 3) and D10 (n = 3) following Cre, along with input control. c Quantification of gel intensities in b, normalized to input and expressed as a percentage of total FlipArray abundance. d Detection and quantification of Cre-mediated inversion of the crRNA FlipArray at the RNA transcript level using RT-PCR (n = 2 infection replicates). The expression of the inverted FlipArray was assessed at multiple timepoints following EFS-Cre infection using sequence-specific primers for the inverted FlipArray transcript as normalized to the Cpf1 mRNA level. e, f Representative Illumina targeted amplicon sequencing of the crNf1 target site (e) and crPten target site (f) in uninfected controls. g, h Representative Illumina targeted amplicon sequencing of the crNf1 target site (g) and crPten target site (h) 7 days after infection with lentivirus containing EFS-Cpf1-puro; U6-NPF-FlipArray. Where relevant, the top 5 most frequent variants are shown, with the associated variant frequencies in the boxes to the right. i, j Representative Illumina targeted amplicon sequencing of the crNf1 target site (i) and crPten target site (j) 17 days after infection with lentivirus containing EFS-Cpf1-puro; U6-NPF-FlipArray and 10 days following EFS-Cre infection. Where relevant, the top five most frequent variants are shown, with the associated variant frequencies in the boxes to the right. k Dot plot detailing the total variant frequencies at the crNf1 and crPten target sites in uninfected cells (red), 7 days after FlipArray transduction (−Cre) (green), and 17 days after FlipArray transduction (+Cre) (blue). n = 2 cell replicates for uninfected group, n = 3 for other conditions. All error bars are mean ± s.e.m