Animal Bytes

Animal Bytes examines biosafety challenges posed when conducting work with animals and provides solutions that promote both safe and responsible research. Good safety and animal husbandry are essential for good science. Learn about best practices when working with animals and applied safety information that can be used every day. Please e-mail any comments, suggestions, or insights to Barbara Johnson at [e-mail].

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats)–Based Gene Drive Technology/Systems

Gene drive systems are used to create modifications in an organism’s genome in such a way that it is more efficiently spread through offspring population than would normally be predicted by Mendelian inheritance. CRISPR-Cas9 technology enables gene drive and is inexpensive, easy to use, and so powerful it has been referred to as “the biggest game changer to hit biology since PCR (polymerase chain reaction).” ¹ Gene drive systems have great potential in gene therapy, the creation of insect vectors that are no longer competent to transmit disease, and the generation of agricultural products that are heartier, nutrient rich, and less perishable. Concern is also justifiable that organisms modified using CRISPR-Cas9 and future versions of the current system may cause irreparable harm to the environment, flora, fauna, and possibly humankind if released into the environment whether by accident, intentionally with approval, or maliciously.^2,3 Gene drive technology is also being used to develop animal models to better study human diseases and therapeutic interventions. ⁴ This poses safety risks to animal handlers and husbandry challenges associated with working with a less familiar species as opposed to more conventional laboratory species such as rodents. Whether conducting work with viral vectors, pathogens, or gene drives, there are also safety concerns regarding the potential for unintentional laboratory exposures. ¹ The ethics and risks of use in humans and human germline material are a prominent topic of discussion but will not be reviewed in this column. ⁵

Safety Concerns

Environmental Impact

Gene drive systems have the ability to rapidly change populations because the system makes changes to both chromatids of the chromosome, not just one chromatid. The result is an organism that is homozygous for the introduced trait, not heterozygous. When a homozygous organism breeds with a wild-type organism, which may or may not bear the trait, it (theoretically always) passes one set of the altered gene(s) to the progeny. This results in more rapidly facilitating the spread of the trait through the population. Using gene drive, it is possible to create an insect that carries a gene that results in sterile progeny. ⁶ On the surface, it may seem appealing to create and release into nature a mosquito or tick that is normally the vector of a human disease that, after a number of generations of breeding with the wild-type vector, results in geographic (or total) eradication of the species, thus preventing the spread of disease. However, the reality is that the net effect on the ecosystem is not well understood, and eradication of an organism that is part of the food chain could have a negative impact, disrupting species further up the food chain, or could allow for a competing, potentially more dangerous, or less desirable species to fill the void. Researchers are focusing on an alternative approach for making changes to vector genomes that prevents the vector from becoming infected, hence stopping disease transmission to humans while posing potentially less risk to the ecosystem. ⁷

Animal Models

Development of animal models to study behavior, disease progression, and the efficacy of vaccines and therapeutics is typically a long and expensive process. Characteristics considered in the use and development of animal models include how closely they mimic humans for the research projects of interest, homogeneity within the population, husbandry and economics, generation time, and ethics. The ability to genetically change a species through breeding, germline modification (eg, transgenic and knock-out/knock-in mice), and other techniques has led to a variety of rodent strains that have become a mainstay of biomedical research. ⁸ Using gene drive tools, researchers are expanding from rodent models to animals that are less commonly worked with in the research setting to include the marmoset, a small nonhuman primate, which is more similar to humans with regard to some behavior, neurologic, and endocrine aspects than currently used rodent species. Kishi et al ⁴ are using gene drive systems to develop marmosets for studying Rett syndrome and other neurodegenerative disorders, while Patrick Hsu at the Salk Institute in La Jolla, California, is reportedly looking at ways to use gene drive to study Alzheimer and Parkinson diseases. Marmosets are noted for becoming acclimated to handling when started at an early age but can still inflict a serious bite as adults and, unlike most small rodents, as adults are worked with using added hand protection such as leather gloves. Husbandry practices (housing size, enrichment, colony aspects, etc) differ from those of common rodent species, requiring vivarium adaptation and staff training.

Possible attempts at using gene drive to manipulate animal models, such as ferrets becoming more susceptible to infection with influenza virus or more capable of spreading infection (super-spreaders), require thought regarding appropriate safety measures. These include primary and secondary containment, occupational health, personal protective equipment, and waste and material inactivation, as well as review by appropriate institutional committees, establishment of a national policy, and institution of a competent regulatory authority with the means to verify that entities conducting the work meet the appropriate standards and capabilities.

Accidental Personnel Exposure

If a goal of using gene drive technology is to introduce a gene into one species to better mimic and study disease or pathology in humans, does this pose a risk to the laboratorian developing the gene drive or introducing it to the target species? The point was raised that an error in the design of CRISPR guide RNA could result in creating a gene drive capable of inducing the pathology (to be studied) in an accidently exposed researcher. ¹ In the case of creating a ferret that is more susceptible to or capable of spreading influenza, what would be the impact of a human laboratory exposure to the gene drive or to the virus from an infected ferret? We have to consider the effect on the exposed worker and the potential for subsequent community spread. With all rapidly evolving technologies and applications, at the outset there are often more questions than definitive answers. Whether all researchers fully appreciate potential hazards associated with gene drive work and can conduct a sufficiently thorough risk assessment to mitigate the hazards is unknown. Similarly, as gene drive technology and its applications are moving so rapidly, the question can be asked as to whether institutional biosafety committees (IBCs), animal care and use committees (ACUCs), and other oversight entities are fully prepared to conduct a comprehensive hazard analysis.

Starting Places for Gene Drive Safety

A few good resources are available in open-source literature that provide a starting point for strategies to safely conduct work using gene drives. The Wyss Institute of Harvard University has published a risk management plan for emerging biotechnology that addresses biosafety and biosecurity and considers whether the technology has dual-use application. ⁹ It describes how research is reviewed before it begins and periodically throughout the project by various committees, such as the IBC, ACUC, institutional review board, and others as appropriate. Approaches to enhancing biosafety and biosecurity are accepted methods for risk reduction employed by industrial hygienists and biosafety professionals and include substitution using materials of lower risk, use of enhanced biosafety levels for containment and personnel protection, committee review of the protocol and review of emerging findings during the project, and determination of the most appropriate means to communicate findings, particularly if there are dual-use implications. The strategy Harvard has outlined has also been described and elaborated upon in further detail in 2 US government publications focused on the conduct of dual-use research of concern (DURC).^10,11 While not all experiments using gene drive constitute DURC, considering DURC biosafety principles and practices could be relevant in bolstering gene drive biosafety.

Risk management with regard to gene drive usage in the environment is of significant concern, and recommendations have been made by Oye et al. ² Concerns include unintended ecological consequences of changing the phenotype or survivability of a given population and whether the alteration introduced to the targeted population could spread to nontarget populations (ie, related species), introducing additional types of unanticipated environmental effects. The uncertainty of how the introduced genetic change in the target population affects the environment is not readily predictable, and if it cannot be reversed or stopped, damage could be permanent and catastrophic. The authors also point to regulatory gaps in gene drive and emerging biotechnology, in part because no single agency has overall responsibility for review and comment. A partial list of the recommendations made by the authors to manage or reduce risk includes the following:

Evaluate the efficacy of reversal drives, which would abrogate the original gene drive change.

Use multiple levels of molecular containment to reduce the risk that drives will spread through wild populations during testing.

Conduct initial field tests in geographic areas devoid of the wild target population.

Test drives in a contained setting mimicking the environment where it may be released.

Monitor the prevalence of released drives by environmental sampling.

Conduct an integrated risk-benefit analysis to include potential for intentional misuse to determine whether or how to proceed with the gene drive research.

Akbari et al ¹² recommend using at least 2 of the “potentially stringent confinement strategies” to bolster gene drive safety. They describe the confinement strategies as falling into 4 categories: molecular, ecological, reproductive, and barrier. Several examples of containment strategies described include the following:

Drives target sequences in laboratory species but are absent from wild populations (molecular).

Laboratory experiments are performed out of the habitable range of the wild-type organism (ecological).

The laboratory strain used cannot reproduce with wild-type organisms (reproductive).

A triple container, anesthetizing insects before opening containers, and one investigator conducting all experiments and accounting for each organism are included (barrier).

During the recent Harvard-Yale IBC Symposium, ¹³ common safety recommendations began to emerge for biosafety professionals and principal investigators. Dr Esvelt suggested, “If you answer ‘yes’ to the following three questions, you are performing a gene drive experiment and must consult with your IBC to ensure that appropriate intrinsic and extrinsic controls are in place.” These questions included the following:

Will the experiment make transgenic, sexually reproducing organisms?

Is an entire CRISPR system encoded in a single DNA construct?

Could it self-insert into the genome?

Symposium participants also discussed questions that should be asked by the IBC and questions to identify the potential for risk to individuals working with gene drive systems. The questions were summarized and amended to include the following:

Does your research involve CRISPR or other gene editing technology?

If yes, please indicate which technology (CRISPR-Cas9, zinc finger nucleases, TALENS, meganucleases, other).

For CRISPR systems, is the guide RNA and nuclease on the same plasmid, vector, or delivery vehicle?

Can this plasmid, vector, or delivery vehicle transfect or infect a human cell?

For CRISPR research involving viral vectors, has a genome target scan (or similar scan) for off-target effects by the guide RNA been completed? This is helpful in assessing the risk of potential exposure in the event of an incident.

These approaches make sense in the context of safeguarding the environment. Additional safeguards in preventing human exposure to gene drives and their payload start by conducting a risk assessment that takes into account the answers to the questions proposed above, as well as the effect of the introduced gene, consequences of erroneous insertion of the gene drive, therapy or reversibility should exposure occur, protective measures to prevent exposure, and substitution of hazardous procedures to reduce the potential for exposure. Thought should also be given to whether novel animal models would provide more valuable insight in biomedical studies. The ability to use gene drive systems to create a new animal model does not mean it will be a better predictor of a disease or therapy in humans and, as mentioned, introduces risk to animal handlers and researchers due to lack of familiarity with the new species and other inherent hazards.

CRISPR-Cas9 and next-generation gene drives provide tremendous research potential and power but, like all new technologies, also bear unknown risks. Safety and security risk assessments and countermeasures will need to take into account unknown risks associated with exposure or release. Assessments should be reviewed at the institutional level and where applicable by federal oversight entities, with outcomes clearly communicated to those involved with the project and countermeasures adhered to carefully. Depending on the nature of the project, researchers should also have a plan in place to communicate effectively with the community before the project starts and possibly at milestones throughout its conduct or the results.