Figure 5.

Installation of diphtheria toxin (DT) resistance mutations in hDTR by miniAGBE-4 in HEK293 cells. (A) Workflow of procedure for mutating the hDTR under DT-based selection via miniAGBE-4 in HEK293 cells. NGS, next generation sequencing. (B) Schematic shows the target sites of AGBE-based sgRNA library for hDTR mutation screening. Coloured boxes indicate boundaries of exon region. Gray lowercase letters represent intron sequences. Black (forward) and yellow (reversed) half arrows targeting the below letters indicate protospacer sequences and direction of individual sgRNA. (C) Images of HEK293 cells with DT treatment for 5 days after miniAGBE-4 editing with indicated sgRNAs. NC, HEK293 cells electro-transfected with miniAGBE-4 only. WT, HEK293 cells electro-transfected with resuspension buffer only. +DT, cultured in DT-supplemented medium. -DT, cultured in normal medium. The effect of individual sgRNA had been verified by three biological replicates. Scale bar: 200 μm. (D) Frequencies of hDTR mutations in DT-resistant cells after miniAGBE-4 with sgRNA-1 or sgRNA-2 editing. Gray values in right represent frequency of mutations. Mutations in hDTR sgRNA-2 targeted splice site (5′-GT) resulting in RNA alternative splicing events. Dark blue values in sgRNA-2 represent scores of the WT and predicted emerging splice donor sites by http://wangcomputing.com/assp/. The black arrow indicates boundary of exon and intron sequences. Target DNA sequence and amino acids (blue and green), PAM (underline), mutant sites and amino acids (red). Splicing defect means the splice site is destroyed. Alt. isoform/cryptic donor means the alternative isoform or cryptic splice site may be activated.