pQS-idgS鏈霉菌基因編輯質粒

Schematic illustration of the CRISPR/Cas9 system and flow chart of one-step dual-function chromogenic screening for positive exconjugants and unmarked mutants. a Scheme for the CRISPR/Cas9-mediated genome editing method. The sgRNA-Cas9 complex cleaves the double-strand DNA proximal to a PAM site, generating a double-stranded DNA break. The double-stranded DNA break is repaired via homologous recombination using a donor template. Horizontal blue arrows indicate PCR primers used to distinguish WT and double crossover progeny. b Maps of the pQS-gusA and pQS-idgS plasmids. The backbone of both plasmids is rep multicopy replicon of plasmid pIJ101, scas9 is controlled by the thiostrepton inducible tipA promoter, the sgRNA cassette is under control of the ermE* promoter, apramycin serves as the selection marker. These plasmids can shuttle between E. coli and actinomycetes. The reporter gene for plasmid curing in pQS-gusA is gusA, under the control of tipA, while idgS, under the control of kasOP*, works in pQS-idgS. c Schematic illustration of time-saving methodology with pQS system and flow chart of the chromogenic screening. The design and construction of genome editing plasmid could be completed in 5 days with the optimized cloning method. During the conjugation process, positive exconjugants could be obtained fast and easily with chromogenic screening. After conjugation and cultivation at 30 °C, exconjugants were obtained. From the chromogenic screening, positive exconjugants containing editing plasmids (pQS-gusA- or pQS-idgS-derived plasmids) were picked and verified. The verified mutants were then cultivated without any antibiotics and diluted to isolate individual colonies. Subsequently, unmarked mutants were obtained quickly with the one-step chromogenic screening system. Finally, further confirmation was performed by PCR validation, Sanger sequencing, and metabolic analysis. pQS-idgS-derived colonies were cultivated on plates 4, 5, and 8. pQS-gusA-derived colonies were cultivated on plates 1, 2, 3, 6, and 7. During the positive exconjugants screening process, non-blue colonies which emerged on plates 2 and 4 were sensitive to apramycin, indicating they were false-positives

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