Enrique Asin-Garcia ^1,2* Maria Martin-Pascual ¹ Claudia de Buck ¹ Max Allewijn ¹ Alexandra Müller ¹ Vitor Martins Dos Santos ^1,2,3*

• ¹ Laboratory of Systems and Synthetic Biology, Wageningen University & Research, Wageningen, Netherlands
• ² Bioprocess Engineering Group, Wageningen University & Research, Wageningen, Netherlands
• ³ LifeGlimmer GmbH, Berlin, Germany

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.