Figure 3.

Amino acid alignment using sequences of mutants and WT plants. (A) When the deletions on the Cas9 cleavage (red arrow) cause stop codon formation on the 4-15’s CsaMLO1 139th amino acid; the stop codon formation was detected on 2-18 and 4-21 CsaMLO1’s 140th amino acid (3rd exon). The Alanine (A) was altered with Asparagine (N) at the 137^th amino acid as a result of the NHEJ caused by Cas9 cleavage at the gRNA1 target. (B) 2 bp deletions cause the unexpected stop codon formation on the 33^rd amino acid of CsaMLO8 in 4-11 and 4-14. The target site of gRNA2 was on the first exon for CsaMLO8 and the changed amino acid structures are at the Cas9 cleavage (10^th aa) site for this gene. The early stop codon formation caused the loss-of CsaMLO8 function in 4-11 and 4-14. (C) The mutations seem to be responsible for the stop codon formation in CsaMLO11 and stop codon’s positions of 4-11 and 4-14’s CsaMLO11 closer to that gRNA3 than 4-15 and 2-18 CsaMLO11. However, ORF shifts are initiated at gRNA3 position in whole CsaMLO11 ^del.