Fig. 3
From: A rapid and efficient strategy for combinatorial repression of multiple genes in Escherichia coli
The components of the multi-gene combinatorial repression system. (A) The schematic of three-sgRNA expression plasmid p3gRNA-LTA and TFs expression plasmid pdCas9-3TFs. The TFs lacIAM (red circle), tetR (yellow circle) and araC (red circle) expressed by pdCas9-3TFs regulate the expression of sgRNA from the promoters of PlacO1, PLtetO−1 and ParaBAD, respectively. The red, yellow and blue squares indicate the recognition sites of BbsI, BsaI and SapI, respectively. The red, yellow, and blue crosses indicate the cleavage sites of BbsI, BsaI and SapI, respectively; (B) The schematic of one-step three-sgRNA ligation strategy based on Golden Gate Assembly. Type IIs restriction endonuclease cleaves p3gRNA-LTA to form sticky ends. Spacers with sticky ends are formed by annealing single-stranded oligonucleotides. T4 DNA ligase (green circle) connects spacers to sites with the same sticky ends. The process can be recycled and enables joining of three sgRNA sequences in the same reaction mixture