이대희 (한국생명공학연구원, 과학기술연합대학원대학교(UST)) | 한빛사논문 > 한빛사

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한국생명공학연구원, 과학기술연합대학원대학교(UST)

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Nucleic Acids Res., 13 December 2022, gkac1104 | https://doi.org/10.1093/nar/gkac1104 CRISPRi-based programmable logic inverter cascade for antibiotic-free selection and maintenance of multiple plasmids

Seong Keun Kim ^{1 2}, Haseong Kim ^{1 2}, Seung Gyun Woo ^{1 2}, Tae Hyun Kim ^{1 2}, Eugene Rha ¹, Kil Koang Kwon¹, Hyewon Lee¹, Seung-Goo Lee ^{1 2}, Dae-Hee Lee ^{1 2 3}

¹Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea.
²Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology, Daejeon 34143, Republic of Korea.
³Department of Integrative Biotechnology, College of Biotechnology and Bioengineering, Sungkyunkwan University, Suwon-si, Gyeonggi-do 16419, Republic of Korea.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
To whom correspondence should be addressed. : Seung-Goo Lee, Dae-Hee Lee

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.

논문정보

형식Research article
게재일2022년 12월 (BRIC 등록일 2022-12-30)
연구진국내 연구진
분야 생명과학 > 생물공학

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