Versatile CRISPR Tools for Gene Regulation

The counterselective gene upp was PCR (2×T5 Super PCR Mix, TSINGKE) amplified from the genome of E. coli MG1655 and adapted to upp^6tgg. The upp variant was then cloned into the low-copy pSC101 vector with the P23119 promoter and NGG PAM by Gibson cloning. The negative control plasmid pTTT-UPP^6TGG-MKATE was modified from pNGG-UPP^6TGG by replacing the PAM sequence from NGG to TTT, and expressing an additional red fluorescent protein (Mkate). The PAM library template plasmid pUPP^6TGG-AmpR was derived from pNGG-UPP^6TGG by co-expressing with the ampicillin resistance gene AmpR using p23119 promoter. The BE elements SpdCas9-CDA, xdCas9 3.7-CDA and dCas9 NG-CDA were commercially synthesized by GENERAL BIOL and cloned into the oriRγ plasmid pHK with Ptet promoter. The sgRNA sequence targeting upp^6tgg was inserted into p15A-gRNA via golden gate assembly (Su et al., 2016 (link)). The sgRNA sequence targeting genomic lacZ gene was inserted into pgRNA-bacteria (Qi et al., 2013 (link)) by Gibson cloning. To validate the repression of GFP using various PAM sequences, SpdCas9 was cloned into pBAD24, generating pdCas9. A total of 49 6tgg-GFP expression plasmids containing TTTT, NNGG, GTGT or GTGC PAMs were constructed by Gibson cloning. All the plasmid constructed were confirmed via sanger sequencing. DNA sequences of the core elements used in this study were listed in Table S1.