Fig. 4: Base editing with engineered Cas9d-MG34-1 and HEARO MG35-1 nickases.
From: Compact Cas9d and HEARO enzymes for genome editing discovered from uncultivated microbes
a Diagrams of base editor constructs with total amino acid length (not to scale). b–d Base editing at multiple genomic target loci. Base editing in E. coli with ABE-MG34-1 (b) and CBE-MG34-1 (c) vs. reference SpCas9 base editors at four target loci. d Base editing in human HEK293T cells with an ABE-MG34-1 (nickase fused with TadA*(8.8 m) deaminase) at three target loci. The target sequence for each locus in b–d is shown above each heatmap. Expected edit positions are represented on the sequence by a subscript number and at each position on the heatmap (squares). Heatmaps in b–d represent the percentage of NGS reads supporting an edit at each position. Values in b and c represent the mean of two independent experiments, while values in d represent the mean of three independent biological replicates. e E. coli survival assay. E. coli was transformed with a plasmid containing the nMG35-1-ABE, a nonfunctional chloramphenicol acetyltransferase (CAT H193Y) gene, and a sgRNA that either targets the CAT gene (target spacer) or not (non-target spacer). Left: E. coli survival under chloramphenicol selection is dependent on the ABE base editing the nonfunctional CAT gene to its wild-type sequence. Right: Diagram showing the target sequence with the nuclease’s required TAM. The “A” base at position 17 from the TAM is expected to edit to “G” to revert the tyrosine residue to histidine and restore chloramphenicol (cm) resistance. f Transformed E. coli was grown on plates containing chloramphenicol concentrations of 0, 2, 3, 4, and 8 ug/mL. Plates also contain 100 ug/mL Carbecillin and 0.1 mM IPTG. Experiments were performed in duplicate.
