Figure captions
Figure 1 The CRISPR/Cas9n (D10A)-mediated base-editing system in S. oneidensis MR-1. Strategy (a) and design (b) of the pCBEso system enabling effective conversion of C to T are shown. APOBEC1-Cas9n (D10A), a fusion protein containing the cytidine deaminase rAPOBEC1 and the Cas9 nickase (D10A); CmR, a chloramphenicol-resistance marker; ColE1, a replication origin for both of E. coli and S. oneidensis ; LacI, Lac repressor; I-SceI, the counter-selectable marker for plasmid curing after base editing. (c) Workflow of the base-editing system in S. oneidensis MR-1. Two BsaI sites were introduced into the pCBSso plasmid. After transforming the targeted pCBEso plasmid into S. oneidensis MR-1 by electrotransformation, the resulting cells were plated on the LB agar plates containing 10 µg/mL chloramphenicol. Colonies on the plates were screened and verified by PCR amplification and Sanger sequencing respectively.
Figure 2 Characteristics of the pCBEso system in S. oneidensis MR-1. (a) Seven 20-nt protospacers containing each NC motif at different positions were selected for evaluating the editing efficiency of the target C. The base editing window of the pCBEso systemin vivo is highlighted by gray shading. (b) Positional influences of each NC motif on base editing efficiency in vivo . The editing efficiencies of each NC motif within the base editing window are presented in a histogram. PCR products of the targeted regions in eight to ten colonies were subjected to Sanger sequencing. The optimal positions of target C within the base editing window are shown in yellow box.
Figure 3 Cytidine editing activity of the pCBEso system targeting an exogenous GFP-lacZ -fused reporter in S. oneidensis MR-1. (a) Sanger sequencing of the targeted region within GFP-lacZ -fused reporter. The protospacer is shown in red wireframe, and amino acids with their codons are indicated. PAM sequence is highlighted in red bold font. The number in red indicates positively edited clones, while the number in black represents total sequenced clones randomly picked from the plates. (b) Flow cytometry analysis of base editing activity of the pCBEso system. The wild-type and MR-1/GFP-lacZ strains were used as negative (GFP-) and positive (GFP+) controls respectively. (c) Editing efficiency of the target C was further confirmed by chromogenic test with X-gal.