Figure 2.

Precise editing of the PPX1 gene. A, Schematic of the PPX1 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 PPX1 ssODN, is expected to introduce two base pair changes (highlighted in yellow) into the genome (bottom sequence). B, Cells co-transfected with the CRISPR/Cas9 (PPX1) RNP and ssODN donor DNA were spread on TAP agar plates containing oxyfluorfen. A representative plate shows two green oxyfluorfen resistant colonies as a consequence of PPX1 gene editing. C, Representative sequencing chromatograms of the WT, fully edited (9×, containing both G→A and C→T changes) and, likely, partly edited (5×, containing only the G→A change) colonies. The chromatogram of an apparently mixed colony (1×), displaying a heterozygous (i.e. WT and fully edited) sequence, is also shown. Figures followed by an X indicate the number of colonies of each DNA type examined by sequencing.