Environment signal dependent biocontainment systems for engineered organisms: Leveraging triggered responses and combinatorial systems

Abstract

As synthetic biology advances, the necessity for robust biocontainment strategies for genetically engineered organisms (GEOs) grows increasingly critical to mitigate biosafety risks related to their potential environmental release. This paper aims to evaluate environment signal-dependent biocontainment systems for engineered organisms, focusing specifically on leveraging triggered responses and combinatorial systems. There are different types of triggers-chemical, light, temperature, and pH-this review illustrates how these systems can be designed to respond to environmental signals, ensuring a higher safety profile. It also focuses on combinatorial biocontainment to avoid consequences of unintended GEO release into an external environment. Case studies are discussed to demonstrate the practical applications of these systems in real-world scenarios.

Keywords: Biocontainment; Combinatorial systems; Engineered organisms; Genetic circuits; Kill switches; Synthetic biology; Triggered responses.

Graphical abstract

Fig. 1

Methods of chemically induced Kill Switches. a. Deadman and Passcode kill switches. b. Toxin-Antitoxin system. c. Promoter engineering techniques. d. Chemically induced dimerization to create an artificial non function hetero dimer protein that is nonfunctional. e. Chemically inducible CRISPR defense system to prevent CRISPR genome editing.; TF, Transcription factor; RBS, Ribosome binding site; CDS, coding DNA sequence; TER, Terminator; GFP, Green fluorescent protein.

Fig. 2

Key innovations in light-responsive gene-editing systems include: a) photoactivable actuator, This is a schematic overview of a Two-hybrid light-inducible gene expression systems is presented. These systems depend on light-triggered interactions between protein 1 (IP) and protein 2 (Photosensor). The IP is fused to a DNA-binding domain (DBD) that specifically binds to its corresponding DNA sequence. The photosensor is attached to a transcriptional activation domain (TF). When light activates the interaction between P1 and P2, TF is recruited to the promoter, leading to the expression of the gene of interest (GOI). b). Engineered photoswitches called Magnets in a Cas9 system, Methods for conditional control of Cas9 activity include various strategies. (A) One approach involves inactivating Cas9 by fusing it to a small molecule- or light-responsive domain, or by splitting Cas9 into N- and C-terminal fragments that can be reassembled in response to light or a small molecule, restoring its activity. In this system, the N- and C-terminal fragments of Cas9 are linked to engineered light-responsive domains, termed positive magnet (pMag) and negative magnet (nMag). When exposed to blue light, pMag and nMag dimerize, creating a split system that results in lower background activity and a greater fold induction of Cas9 activity. and C. The engineering of single-chain photoswitchable Cas9 (ps-Cas9) proteins these can be implemented individually or together as a biocontainment method. A strategy for degrading Cas9 involves using a heterobifunctional small molecule, where one end binds to a small-molecule binding domain attached to Cas9, while the other end targets CRBN. This interaction promotes ubiquitination and subsequent proteasomal degradation of Cas9.