AUTHOR=Asin-Garcia Enrique , Martin-Pascual Maria , de Buck Claudia , Allewijn Max , Müller Alexandra , Martins dos Santos Vitor A. P. TITLE=GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida JOURNAL=Frontiers in Bioengineering and Biotechnology VOLUME=12 YEAR=2024 URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2024.1426107 DOI=10.3389/fbioe.2024.1426107 ISSN=2296-4185 ABSTRACT=

Synthetic genetic circuits have revolutionised our capacity to control cell viability by conferring microorganisms with programmable functionalities to limit survival to specific environmental conditions. Here, we present the GenoMine safeguard, a CRISPR-Cas9-based kill switch for the biotechnological workhorse Pseudomonas putida that employs repetitive genomic elements as cleavage targets to unleash a highly genotoxic response. To regulate the system’s activation, we tested various circuit-based mechanisms including the digitalised version of an inducible expression system that operates at the transcriptional level and different options of post-transcriptional riboregulators. All of them were applied not only to directly control Cas9 and its lethal effects, but also to modulate the expression of two of its inhibitors: the AcrIIA4 anti-CRISPR protein and the transcriptional repressor TetR. Either upon direct induction of the endonuclease or under non-induced conditions of its inhibitors, the presence of Cas9 suppressed cell survival which could be exploited beyond biocontainment in situations where further CRISPR genome editing is undesirable.