Figure 6.

Precise editing of the ALS1 gene. A, Schematic of the ALS1 gene showing the target (highlighted in bluish purple) and reverse complement PAM (highlighted in gray) sequences. A black arrowhead indicates the Cas9 cleavage site. HDR of the DSB, using as template the transfected ALS1 ssODN, is expected to introduce two base pair changes (highlighted in yellow) into the genome (bottom sequence). These sequence changes cause an amino acid substitution (K257T) in the encoded ALS enzyme and create a new EcoRV restriction enzyme site (underlined in black). B, DNA sequences of SMM resistant colonies showing alterations at the ALS1 target site relative to the WT. Base substitutions are highlighted in yellow. Figures followed by an X (i.e. 45× and 23×) indicate the number of colonies of each DNA type analyzed by sequencing (complete sequences for each colony are shown in Supplemental Figure S11). Other numbers indicate the colony names.