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.