# Towards the development of a CRISPR-Cas9 based kill switch for Saccharomyces cerevisiae

## TL;DR

This paper introduces a CRISPR-based kill switch for yeast cells, showing how it can inhibit growth but still needs improvement to fully kill cells.

## Contribution

The study presents the first CRISPR-based kill switch for Saccharomyces cerevisiae and identifies ways to improve its effectiveness.

## Key findings

- Inducing CRISPR targeting of essential genes in yeast causes growth inhibition.
- Mutations in gRNA and target genes lead to system inactivation and cell escape.
- Simultaneous expression of two gRNAs improves the kill switch's performance.

## Abstract

Advancements in synthetic genetic circuits have enabled programmable and condition-dependent control of microbial cell growth. CRISPR-Cas9-based kill switches, genetic systems that program cells to lose viability in response to specific conditions, have recently been demonstrated for bacterial cell factories but not yet in yeast.

In this study, we present a foundational demonstration for a CRISPR-based kill switch in Saccharomyces cerevisiae, CRISPR KiSS. The CRISPR KiSS employs inducible CRISPR targeting essential genes to elicit growth inhibition. The activation of the KiSS system is achieved through conditional expression of a guide RNA (gRNA) upon anhydrotetracycline (ATc) induction, thereby activating CRISPR-mediated gene disruption. We demonstrate that targeting the essential genes (ERG13, PGA3, TPI1 or CDC19) leads to severe growth inhibition upon ATc induction. Still, the current set up does not allow complete killing of the cells due to system inactivation, e.g. escape from CRISPR based cutting. We studied reasons for system inactivation and substantially improved the system by simultaneous expression of two different gRNAs. Sequencing escape mutants revealed mutations in both the gRNA sequences and target genes as potential sources of system inactivation.

This work highlights the potential of harnessing a CRISPR-based kill switch in S. cerevisiae. Cells expressing the system were able to escape growth inhibition through mutations and further optimization of the KiSS system is still needed for it to be used in various cell factory applications.

The online version contains supplementary material available at 10.1186/s12934-026-02959-2.