Appendix C

Questions to Stimulate Consideration of Framework Factors

The following illustrative questions were developed to stimulate consideration of the framework factors and facilitate use of the framework to assess specific potential capabilities. These are not intended to represent every question that can be posed, and some questions can be applicable to assessing more than one factor.

Usability of the Technology—Ease of Use

• How long is the oligonucleotide, gene, or genome involved?
• If an entire genome is being created, how easy is it to assemble?
• For an entire genome, how easy is it to “boot”?
• What is the scale and complexity of modification or synthesis involved? For example, is the target a virus, bacterium, fungus, or a larger organism, and how does this affect the ease of use?
• Can the desired construct be ordered commercially, or would regulatory oversight (e.g., Select Agent rules) or construct length make this unlikely?
• Are reagent kits available to make the process easier?
• Are genomic design tools and relevant “parts” databases available to help achieve desired goals?
• How reliable is the available genomic sequence information?
• How reliable is the available genotype-to-phenotype information, and how does this affect the ease of use for the intended purpose?
• Is there a recipe or standard operating procedure available for the intended use, and if so, has it been demonstrated to work previously?
• Is specialized equipment required, and if so, is it readily available for purchase or via contract?
• What level of specialized knowledge, hands-on training, and tacit knowledge is required?
• Are suitable test conditions (e.g., cell cultures, model organisms) available?

Usability of the Technology—Rate of Development

• Are significant improvements to the technology being published on at least an annual basis?
• What aspects are improving? (Examples of aspects to consider include total processing time, cost, laboratory space footprint, level of automation, accuracy, throughput, user interface, and output reporting.)

• What types of uses are driving commercial development and market adoption?
• Is there competition spurring the rate of the technology’s development, or does one company have a monopoly?
• Are there multiple different markets for the technology, spurring technological development and innovation, or is it tightly focused on one specific market?
• Is there an open-source user community helping to drive the technology forward by sharing new developments?

Usability of the Technology—Barriers to Use

• Are there critical bottlenecks that, once overcome, will significantly improve ease of use (e.g., CRISPR/Cas9 for gene editing, photolithography for oligonucleotide synthesis)?
• What barriers may hinder wider market adoption and penetration of the technology involved, and how might these be overcome?
• Would significant improvements in Build capabilities (e.g., capacity for increased construct length or reduced cost of synthesis) be accompanied by corresponding improvements in capabilities for Design and Testing relevant to the intended application, or would those aspects remain as barriers?
• Are there gaps in fundamental knowledge about pathways and genotype-to-phenotype relationships that may hamper the use of genomic design tools for the intended use?

Usability as a Weapon—Production and Delivery

• Could synthetic biology (or its use in combination with other biotechnology advances) be used to enhance replication or growth characteristics of an agent in order to support scale-up?
• Could synthetic biology (or its use in combination with other biotechnology advances) help to scale up production of the agent without its losing infectivity or other key features?
• Could synthetic biology be used to make an agent “hardier” in the varied environments it may encounter during storage and delivery (e.g., could it survive the adverse conditions that might be expected in the context of dispersal)?
• Could synthetic biology be used to stabilize the agent or facilitate dispersal and survival?
• How might the agent be delivered to those targeted (e.g., mass dispersal, contamination of food or water, a needlestick), and how might this delivery mechanism affect requirements for production, stabilization, or testing?
• Could synthetic biology (or its use in combination with other biotechnology advances) facilitate novel or enhanced forms of delivery?
• Is large-scale production of the agent needed to have an impact?
• Could synthetic biology help to reduce the organizational footprint, expertise, or equipment required for production?

Usability as a Weapon—Scope of Casualty

• Could synthetic biology be used to enhance host susceptibility to a given agent in a way that would worsen the severity of an attack or increase the number of casualties?
• How many individuals could be targeted for harm using this capability (ranging from a single assassination to thousands of people, or more)?
• Is the agent highly transmissible, thus allowing it to spread beyond those affected by the initial attack?
• Would an attack based on this capability be expected to be lethal or incapacitating?
• Could an attack based on this capability have psychological effects or affect the functioning of the targeted group? For example, could it incite fear, create panic, and/or allow the takeover of a particular region or infrastructure?
• What might the duration of the impact be?

• In what environment(s) might the agent be used?
• Could the agent become established in domestic animals or agricultural livestock (e.g., plague in cats) or wildlife, causing longer-term effects on humans and requiring difficult and costly eradication?

Usability as a Weapon—Predictability of Results

• Does the agent need to be tested extensively to confirm that it is efficacious?
• Is there a relevant animal model for the agent? How predictable is that model for human infection by the same agent?
• What is the fidelity of the technology? How reproducibly can a particular result be obtained?
• Are there known engineering strategies or preexisting research outlining methods to predictably produce the desired result? Can the properties of a bioagent be modeled with computational tools?
• Is there knowledge regarding the evolutionary stability of an engineered pathogen or pathway? For example, is it likely a synthetic construct will mutate to increase or decrease functionality or activity? Or can slow-evolving pathogens be generated to avoid attenuation?

Requirements of Actors—Access to Expertise

• How common and widespread is the technical expertise needed to exploit the necessary technology, and could expertise in another, related area suffice?
• Would expertise in more than one area be required to pursue the capability, and would the range of technological expertise likely require a group of people to provide the expertise?
• Would developing this capability require or be enhanced by interaction with the legitimate research community, or could it be performed autonomously?

Requirements of Actors—Access to Resources

• What are the equipment costs, and how quickly are equipment costs decreasing?
• Are cheaper versions of the necessary technology becoming available, and are they robust enough to raise concerns?
• Can reagents be acquired from multiple vendors, or is there a secondary market (e.g., eBay) where the equipment can be acquired at a lower cost?
• What are the material or reagent costs?
• What is the shelf life of the required reagents?
• What are the labor costs? Is specialized training required, and if so, what are the costs involved in that training?
• What are the maintenance or service costs, and how frequently is maintenance or service needed?
• What facility costs are associated with the necessary technology (e.g., special plumbing, cooling, airflow, filtration, vibration isolation)?
• What is the biosafety risk to the actor, and what costs might the actor incur to protect the safety of those doing the work?
• What would it cost to conceal the pursuit of this capability from authorities (or other nations)?

Requirements of Actors—Organizational Footprint Requirements

• What is the organizational footprint (e.g., equipment and other laboratory infrastructure, personnel) needed to utilize the necessary technology?
• Is the infrastructure required to use this technology widespread or rare?
• Could existing organizations or infrastructure be leveraged to develop this capability (e.g., dual use of legitimate biotechnology infrastructure), or would the work require a secret facility with a particular set of infrastructure requirements?

• If additional infrastructure would be required for malicious use, would it require an incremental increase in capacity or major additions?

Potential for Mitigation—Deterrence and Prevention Capabilities

• Can the development of this capability be controlled or prevented through regulation or other means, either in the United States or internationally? Do nations have agreements relevant to applicable regulations?
• Is the necessary technology geographically centralized or widely distributed?

Potential for Mitigation—Capability to Recognize an Attack

• To what degree can beneficial and malicious use of the technology involved in this capability be distinguished?
• Are there particular activities or equipment associated with this technology that may indicate when it is being used to prepare for an attack?
• Could the capability be used to engineer an agent that evades typical disease surveillance methodologies (e.g., to cause an unusual constellation of symptoms)?
• Could the capability be used to engineer an agent that evades typical identification and characterization methodologies (e.g., to create an agent that lacks the phenotypes or DNA sequence used for laboratory identification)?
• Would it be possible to assess whether the agent was created synthetically, as opposed to emerging naturally?
• Could the capability enable targeting of particular subpopulations, and if so, could this targeting be detected with available disease surveillance mechanisms?
• Could environmental surveillance (e.g., direct sensing via BioWatch or similar approaches, animal sentinels, sensing without direct contact [standoff detection]) provide earlier warning of a bioweapon attack than waiting for ill individuals to present in the public health system?
• Can mining social media in real time provide indications of when and where an attack or outbreak based on this capability might take place, compared to traditional public health surveillance mechanisms?

Potential for Mitigation—Attribution Capabilities

• How feasible would it be to use DNA sequencing to compare samples of the agent with samples from recovered evidence?
• Would the technique used to construct or modify the agent leave a genomic “scar” that could potentially be used as evidence?
• Would it be possible to identify a design “signature” linking the use of this technology with a given group or laboratory?
• Would the development of this capability be associated with certain physical properties that could be used to compare samples of the agent with samples from recovered evidence?

Potential for Mitigation—Consequence Management Capabilities

• Will existing civilian and military public health infrastructure and mitigation approaches to minimize morbidity and mortality be effective against an attack using this capability?
• Are there currently effective medical countermeasures available for an attack using this capability, or would it be possible to quickly develop vaccines, drugs, or antitoxins to mitigate the spread and impact of the agent over the longer term?
• Would the effectiveness of those mitigation approaches rely on knowing how an agent was created?
• Would it be possible to understand the genotype, phenotype, or chemical composition of the agent to inform how its effect can be mitigated?