Supplementary Figure 1: Rationale and workflow for CRISPR-Cas9 inactivation of common antibiotics.
(A) Pie chart showing the frequency of discovery of different antibiotics from 60 Gram negative active actinomycete extracts. Frequencies are calculated from Cox et al.2 (B) Nucleotide identity (%) is shown across targeted biosynthetic genes in the streptothricin gene cluster. Identity was calculated by alignment of 29 streptothricin BGCs. The locations of target sites are shown by red sgRNAs and regions of 100% sequence conservation are indicated with green vertical bars. (C) Plasmid map of the pCRISPomyces-2 system14. (D) Key steps for a streamlined and generalizable strategy to efficiently inactivate production of a commonly found antibiotic using the pCRISPR-Cas9 system. Once targeting plasmids are constructed, the process can be completed in 30 days with just six days of hands-on work.
