Figure 2.

Design and functional validation of the Rosa26^PE2 prime editor allele.

a. Schematic depicting the design of the Cre-inducible Rosa26^PE2 allele.

b. Schematic depicting the formation of hU6-pegRNA-EF-1α-Cre (UPEC) and hU6-pegRNA-EFS-mScarlet (UPEmS) vectors from templates encoding a red fluorescent protein (RFP) by Golden Gate assembly.

c. Bright-field images of pancreatic organoids derived from chimeric prime editor mice and wild-type mice. With and without treatment with neomycin.

d. Bright-field and fluorescent images showing PE2-P2A-mNG expression only after exposure to Cre encoded by a UPEC vector.

e. Schematic depicting the derivation of multiple organoids and a fibroblast cell line from Rosa26^PE2/+ prime editor mice.

f. Editing efficiency of a trinucleotide (+GGG) insertion located eight base pairs downstream of the start codon in Dnmt1 in pancreatic organoids, lung organoids, and tail tip-derived fibroblasts. Unintended indel byproducts in all conditions were present in <1% of sequencing reads. Data and error bars indicate the mean and standard deviation of three independent transductions.

g. Editing efficiency and indel byproduct frequency of Dnmt1^+GGG in liver tissue one week after tail vein injection with LNPs harboring either Cre mRNA and pegRNA (left) or pegRNA alone (right). Data and error bars indicate the mean and standard deviation of three to five independent mice.

h. Bright-field and fluorescent images of pancreases derived from Rosa26^PE2/+ (left) or Pdx-1 Cre;Rosa26^PE2/+ mice (right).

i. Immunofluorescence imaging of intestinal tissue derived from Villin-CreER^T2; Rosa26^PE2/+ mice that were either untreated (left) or exposed to tamoxifen (right; 4-OHT). Tissue slides were stained with the DNA stain DAPI (4′,6-diamidino-2-phenylindole; top) or with an antibody specific to Cas9 (bottom). Scale bar indicates 100 μm.