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CRISISS: A Novel, Transcriptionally and Post-Translationally Inducible CRISPR/Cas9-Based Cellular Suicide Switch
Maximilian Amberger1, Esther Grueso1, Zoltán Ivics1
1Research Center, Division of Hematology, Gene and Cell Therapy, Paul-Ehrlich-Institute, 63225 Langen, Germany.
Abstract:
With the ever-increasing developing rate of gene and cellular therapy applications and growing accessibility due to products receiving regulatory approval, the need for effective and reliable safety mechanisms to prevent or eliminate potentially fatal side effects is of the utmost importance. In this study, we present the CRISPR-induced suicide switch (CRISISS) as a tool to eliminate genetically modified cells in an inducible and highly efficient manner by targeting Cas9 to highly repetitive Alu retrotransposons in the human genome, causing irreparable genomic fragmentation by the Cas9 nuclease and resulting cell death. The suicide switch components, including expression cassettes for a transcriptionally and post-translationally inducible Cas9 and an Alu-specific single-guide RNA, were integrated into the genome of target cells via Sleeping-Beauty-mediated transposition. The resulting transgenic cells did not show signs of any impact on overall fitness when uninduced, as unintended background expression, background DNA damage response and background cell killing were not observed. When induced, however, a strong expression of Cas9, a strong DNA damage response and a rapid halt of cell proliferation coupled with near complete cell death within four days post-induction were seen. With this proof-of-concept study, we present a novel and promising approach for a robust suicide switch with potential utility for gene and cell therapy in the future.
Insights
We developed CRISPR-induced suicide switch (CRISISS) to eliminate genetically modified cells. This system safely targets Cas9 to repetitive DNA, inducing cell death for gene and cell therapy safety.
Area of Science:
- Genetics
- Molecular Biology
- Biotechnology
Background:
- Gene and cellular therapies are rapidly advancing, necessitating robust safety mechanisms.
- Existing safety strategies require improvement for effective elimination of modified cells.
- Controlling genetically modified cells is crucial for therapeutic safety and efficacy.
Purpose of the Study:
- To develop and validate a novel inducible suicide switch for eliminating genetically modified cells.
- To assess the safety and efficiency of the CRISPR-induced suicide switch (CRISISS) system.
- To provide a reliable tool for enhancing safety in gene and cell therapy applications.
Main Methods:
- Engineered a CRISPR-Cas9 system targeting repetitive Alu retrotransposons in the human genome.
- Integrated suicide switch components (inducible Cas9, Alu-specific sgRNA) using Sleeping Beauty transposition.
- Evaluated cell fitness, DNA damage response, proliferation, and cell death upon induction.
Main Results:
- Uninduced transgenic cells exhibited normal fitness with no background effects.
- Induction led to robust Cas9 expression, significant DNA damage response, and proliferation arrest.
- Near-complete cell death was observed within four days post-induction.
Conclusions:
- CRISISS provides an effective and inducible method for eliminating genetically modified cells.
- The system demonstrates high efficiency and safety, with no adverse effects when uninduced.
- CRISISS represents a promising safety tool for future gene and cell therapy development.
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