Figure 3.

Ex vivo prime editing and functional testing of Kras and Trp53 mutations.

a. Editing efficiency and indel byproduct frequency of the Kras^G12D transition mutation (G:C to A:T) templated by a cohort of pegRNAs based on a single Cas9 spacer (n = 3 for each pegRNA). pegRNAs are delineated by differences in the lengths of the primer binding site (PBS) and reverse transcriptase template (RTT). Data and error bars indicate the mean and standard deviation of three independent transductions.

b. Editing activity of four engineered pegRNAs (epegRNAs) templating either the Kras^G12D transition or the Kras^G12C, Kras^G12A, or Kras^G12R transversions in tail tip-derived fibroblasts or pancreatic organoids (Kras^G12D and Kras^G12C). Data and error bars indicate the mean and standard deviation of three independent transductions. epegRNAs were generated by appending the trimmed evopreQ₁ motif after the primer binding site of the leftmost pegRNA depicted in Fig. 1a. Indel byproduct calculations were pooled from all conditions within each tissue.

c. Allele frequencies of Kras^G12D or Kras^G12C mutations in pancreatic organoids before and after two passages of treatment with gefitinib (1 μM) (n = 1). Gefitinib treatment selects for cells containing prime edited Kras^G12D or Kras^G12C mutations.

d. Bright-field images of prime edited Kras^G12C or Kras^G12D organoids treated for four days with either control DMSO, sotorasib (2 μM) and gefitinib (1 μM), MRTX1133 (5 μM), or MRTX1133 and gefitinib.

e. Viability of Kras^G12D pancreatic organoids under various treatment conditions. Viability was quantified using the alamarBlue HS Cell Viability Reagent, which is metabolized into a fluorescent derivative in living cells. Bars represent the range across two independent replicates.

f. Allele frequency of Kras^Y96C in Kras^G12C organoids during and after treatment with sotorasib (2 μM) and gefitinib (n = 1). After two passages, organoids were split into two groups, which included continued treatment (continuous treatment) in one group and removal of treatment in a second group (transient treatment).

g. Allele frequencies of three Trp53 mutations in Trp53^flox/+ pancreatic organoids treated with nutlin-3a for three to five passages after transduction with UPEC vectors. Indel byproduct frequencies are also included. Note that the highest indel frequency depicted for Trp53^R245W derives primarily from a scaffold insertion in a single replicate. Trp53^R245Q and Trp53^R245W are homologous to mutations commonly observed in pancreatic cancer patients (TP53^R248Q and TP53^R248W), as described in Supplementary Figure 8. Trp53^R250FS denotes a dinucleotide deletion that induces a frameshift mutation. Trp53^{M240FS−14nt} denotes a fourteen-nucleotide deletion. Data and error bars indicate the mean and standard deviation of two to five independent transductions.

h. Immunoblot indicating detectable levels of p53 protein in prime edited Trp53^flox/R245Q and Trp53^flox/R245W organoids and an absence of detectable protein in Trp53^flox/R250FS organoids.