Figure 4.

Co-editing of the WDTC1 gene in colonies selected for precise PPX1 editing. A, Schematic of the WDTC1 gene showing the target (highlighted in bluish purple) and PAM (highlighted in gray) sequences. Other symbols are as described under Figure 1A. HDR of the DSB, using as template the transfected WDTC1 ssODN, is expected to introduce four base-pair changes (highlighted in yellow) into the genome (bottom sequence). These sequence changes destroy the WDTC1 start codon and create a new BssHII restriction enzyme site (underlined in black). B, The WDTC1 target region of selected oxyfluorfen-resistant colonies was amplified by PCR (with primers F3 and R3) and the PCR products digested with BssHII. The panels show representative reverse images of agarose resolved PCR products stained with ethidium bromide. The sizes of molecular weight markers are indicated in base pairs. g1, WT strain. C, DNA sequences of oxyfluorfen resistant colonies showing alterations at the WDTC1 target site relative to the WT. Insertions, indicating number of base pairs, are depicted in red. Base substitutions are highlighted in yellow. Complete sequences for colonies exhibiting insertions at the WDTC1 gene are shown in Supplemental Figure S7.