CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids. Issue 22 (13th December 2022)

• Record Type: Journal Article
• Authors: Kim, Seong Keun; Kim, Haseong; Woo, Seung Gyun; Kim, Tae Hyun; Rha, Eugene; Kwon, Kil Koang; Lee, Hyewon; Lee, Seung-Goo; Lee, Dae-Hee
• Is Part Of: Nucleic acids research. Volume 50:Issue 22(2022)
• Page Start: 13155
• Page End: 13171
• Publication Date: 2022-12-13
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkac1104
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: Antibiotics have been widely used for plasmid-mediated cell engineering. However, continued use of antibiotics increases the metabolic burden, horizontal gene transfer risks, and biomanufacturing costs. There are limited approaches to maintaining multiple plasmids without antibiotics. Herein, we developed an inverter cascade using CRISPRi by building a plasmid containing a single guide RNA (sgRNA) landing pad (pSLiP); this inhibited host cell growth by repressing an essential cellular gene. Anti-sgRNAs on separate plasmids restored cell growth by blocking the expression of growth-inhibitory sgRNAs in pSLiP. We maintained three plasmids in Escherichia coli with a single antibiotic selective marker. To completely avoid antibiotic use and maintain the CRISPRi-based logic inverter cascade, we created a novel d -glutamate auxotrophic E. coli . This enabled the stable maintenance of the plasmid without antibiotics, enhanced the production of the terpenoid, (−)-α-bisabolol, and generation of an antibiotic-resistance gene-free plasmid. CRISPRi is therefore widely applicable in genetic circuits and may allow for antibiotic-free biomanufacturing.