PERSPECTIVE https://doi.org/10.1038/s41589-018-0056-x Next-generation biocontainment systems for engineered organisms Jeong Wook Lee 1,2,10, Clement T. Y. Chan3,4,5,6,7,10, Shimyn Slomovic 1 and James J. Collins1,3,4,5,8,9* The increasing use of engineered organisms for industrial, clinical, and environmental applications poses a growing risk of spreading hazardous biological entities into the environment. To address this biosafety issue, significant effort has been invested in creating ways to confine these organisms and transgenic materials. Emerging technologies in synthetic biology involving genetic circuit engineering, genome editing, and gene expression regulation have led to the development of novel bio- containment systems. In this perspective, we highlight recent advances in biocontainment and suggest a number of approaches for future development, which may be applied to overcome remaining challenges in safeguard implementation. major goal of synthetic biology is to design and construct This Perspective article joins other recent reports16–18 in an effort genetically modified organisms (GMOs) with novel func- to capture the many challenges of GMO safeguarding and further tions and biological behaviors that can be used for biomedi- offers directions for overcoming them. Here, the main focus is to A 1–3 cal, industrial, and environmental applications . In addition to discuss how emerging technologies may be harnessed to create new their potential use in technological applications, GMOs serve as biocontainment methods that complement existing systems in order powerful research tools for studying biological processes and prin- to improve the safety of GMO practices. We first detail the general ciples and have become prevalent in many science and engineering requirements of an effective biocontainment system and present a fields. Depending on the purpose, GMO release into the environ- number of synthetic biology approaches that are leading the race ment may be deliberate, such as in waste treatment, or uninten- toward effective GMO safeguarding while discussing their advan- tional. While many GMOs are safe for release, as their engineered tages and limitations. We then outline the latest developments and functions reduce cellular fitness and render the spread of transgenic apply insight gathered from existing approaches to propose strate- material unfavorable, biocontainment measures become necessary gies for building next-generation biocontainment systems. to constrain proliferation when GMOs are able to outcompete nat- ural organisms and negatively affect the environment and human Criteria for effective biocontainment health. Key safeguard requirements must be met to prevent the release and Since the 1970s, scientists and the general public have voiced proliferation of GMOs in natural environments and their ensuing concern that the intentional or accidental release of GMOs may lead interaction with ecosystems. GMOs are living entities that can grow to an unexpected impact on the environment4–6. Although there are and self-replicate; therefore, any embedded biocontainment system no reports of biological hazards caused by GMOs thus far, many must be extremely robust to prevent the release of even a small frac- studies have shown that the use of engineered organisms can result tion of organisms that could grow to dominate an ecosystem. The in the invasion of transgenes into the natural environment7–12. One National Institute of Health has presented a guideline stating that a alarming example regards the spread of antibiotic-resistance genes GMO escapee rate below 1 in 108 cells is considered to be acceptably that are often incorporated into GMOs. A report revealed that resis- safe6, and a number of existing biocontainment systems have met tance genes were found in water polluted by farmland fecal waste this goal. However, with an ever-increasing scale in GMO deploy- and could be transferred to natural bacterial species by bacterio- ment, the containment and/or killing efficiencies of these systems phages13. Such transgene leakage can directly impact human health may not be sufficient to prevent a buildup of GMOs in the environ- by increasing drug resistance among pathogens. ment. Therefore, continuous improvements in system robustness With the rapid rise in the design and deployment of GMOs in and efficiency are needed to ensure biosafety. recent years, the chance of engineered organisms influencing eco- Long-term system stability is another key criterion for effective systems and human health is expected to increase14,15. Therefore, safeguarding, as GMOs are often designed and expected to repli- continuous effort has been invested in designing safeguard measures cate through many generations and different growth phases. To to prevent GMO escape14,16,17 (Fig. 1). The resulting biocontain- maintain robustness and stability, genetic safeguard systems must ment strategies focus on the engineered prevention of self-repli- take into account the potential undermining effects of genetic cation, synthetic auxotrophism, genetic circuit–actuated killing, silencing mechanisms, such as DNA recombination and muta- and horizontal gene-transfer blocking involving a wide range of genesis. Moreover, the biocontainment systems themselves often cellular mechanisms. place a metabolic burden on engineered hosts or cause background 1Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA. 2Department of Chemical Engineering, Pohang University of Science and Technology, Pohang, Gyeongbuk, Republic of Korea. 3Institute for Medical Engineering & Science, Massachusetts Institute of Technology (MIT), Cambridge, MA, USA. 4Department of Biological Engineering, MIT, Cambridge, MA, USA. 5Synthetic Biology Center, MIT, Cambridge, MA, USA. 6Department of Biology, The University of Texas at Tyler, Tyler, TX, USA. 7Department of Chemistry and Biochemistry, The University of Texas at Tyler, Tyler, TX, USA. 8Harvard–MIT Program in Health Sciences and Technology, Cambridge, MA, USA. 9Broad Institute of MIT and Harvard, Cambridge, MA, USA. 10These authors contributed equally: Jeong Wook Lee, Clement T. Y. Chan. *e-mail: [email protected] 530 NATURE CHEMICAL BIOLOGY | VOL 14 | JUNE 2018 | 530–537 | www.nature.com/naturechemicalbiology © 2018 Nature America Inc., part of Springer Nature. All rights reserved. NATURE CHEMICAL BIOLOGY PERSPECTIVE Biocontainment research 1,500 Programmable biocontainment circuits34 Number of publications Synthetic auxotrophy 2,000 examples27,28 First active genetic NIH guideline for Multivector systems for viral First biocontainment example 73 6 22 77 containment biocontainment particle production for clinical use 500 1976 1984 1992 2000 2008 2016 Asilomar First biocontainment First patent for GURT Biocontainment system Multilayered conference4,5 example in bioremediation36 in agricultural use19 to confine plasmids72 biocontainment30,31 Fig. 1 | Biocontainment research timeline. A chart representing the number of published articles found on PubMed using the search terms “biocontainment,” “biosafety,” and “biosecurity”. toxicity, which may limit the desired cellular performance and GMOs that depend on noncoding amino acids (ncAA) to survive. further increase the selective pressure to disrupt the biocontain- In these strains, all naturally occurring amber stop codon sites ment apparatus. To address this problem, safeguard systems must (UAG) have been replaced with an ochre stop codon (UAA). The be tightly regulated when GMOs are under permissive conditions. strains express a non-native tRNA and aminoacyl-tRNA synthetase Lastly, as engineered cells may be required to function in a variety pair that incorporates an ncAA into amber codons that have been of environments for different applications, rapid customization is a strategically placed within the coding sequences of essential genes. beneficial trait. To meet these criteria, various promising biocon- The maintenance of critical cellular activities therefore requires tainment strategies have been developed, as described below. the presence of the ncAA, which is only supplied in the designated environment26,27 (Fig. 2b). To restrict GMOs from circumventing Pioneering biocontainment strategies this mechanism by nonspecifically incorporating natural amino Biocontainment via engineered prevention of self-replication. acids into the amber stop codon, Mandell et al. structurally rede- Some of the earliest examples of biocontainment focused on the signed essential enzymes to depend on the ncAA’s residue for enzy- concept of blocking self-replication in GMOs. One such example, matic activity28. This strict synthetic-molecule dependency leaves Genetic Use Restriction Technology (GURT), was developed in the the resulting auxotrophs with extremely low escapee frequencies late 1990s to restrict the unintended and unauthorized spread of (10−12). Unfortunately, implementing this type of strategy requires engineered plants by disabling their reproductive ability19. Generally, extensive genome editing in the microbial strain, which limits its this approach involves placing seed development genes under the availability. Additionally, synthetic auxotrophy cannot prevent the control of chemically inducible promoters such that when the engi- release of transgenic elements through various biological activities neered crops are grown in open environments the lack of inducing such as horizontal gene transfer. chemicals leads to infertile seed formation. Of note, GURTs