The questions addressed below cover a very wide range of topics. Equally, many key features of the Insect Allies program remain unclear. Consequently, despite our best efforts there may be inaccuracies on this and other pages on this website. We undertake to correct mistakes when we have been made aware of them.
Some of the topics addressed here are complex and that among the 5 authors of the Science article there is not necessarily
- Shared expertise on all points
- Agreement about the nuances and subtleties of every point. Though on the key issues addressed in the Science article there is unanimity
This is actually common to all multi-author articles.
What our article is about
- It is about the immediate biosecurity implications of the Insect Allies program and what the international response to it could be
- It is also a request for the funder of the Insect Allies program to answer how its stated routine agricultural aims can be achieved without a much earlier substantial biological weapons proliferation risk occurring much earlier.
What our article is not about
- It is not about biosafety - though this is a very important issue that will need to be considered
- It is not about similar viral techniques intended to be confined inside buildings e.g. within hospitals or research facilities
- It is not a generic
- It is not simply a call for increased transparency - unless it is combined with a willingness and framework to alter the goals of the Insect Allies program to reflect obvious global concerns
- It is not about potential impacts occurring in 20-40 years - it is about immediate biosecurity impacts that may already have started to happen.
Even as the halfway point of the Insect Allies program approaches, DARPA has chosen not to publicly describe in its response to our Science article what is the basis of their having concluded that a developmental pathway exists that circumvents the early proliferation of biological weapons (described by the black development path in the accompanying image). This is in addition to explaining in detail why their developmental plan is easier to develop than alternative paths (described by the red paths). As our Science article makes clear, this is central to justifying the wisdom of embarking on the development of HEGAAs, and many other types of GM viruses.
Over the next five years, only a minority of anticipated CRISPR-inspired innovations will involve intentional environmental releases (see recent NAS report). HEGAAs, and some other GM viruses, have the property of an early-stage biological weapons proliferation risk that is not shared with most other proposed techniques (including avant-garde ones like gene drive).
Choosing not to clearly address these obvious issues of global concern, as detailed in the Science article, makes their current model of develop first and explain later an especially unwise path, particularly for this insect-delivered program, that in many ways appears to be designed to get carried away.
What are HEGAAs-horizontal environmental genetic alteration agents ?
- HEGAAs are viruses which have been genetically modified to gain a capacity to edit the chromosomes of a target species (e.g. plant or animal) when intentionally released into the environment
- The word “horizontal” comes from their ability to be transmitted in the environment by infection
- The word “environmental” comes from the intention for these genetically modified viruses to be dispersed into the environment.
- The words “genetic alteration agents” comes from the capacity to alter the chromosomes of a target species. This might be through causing a random mutation or introducing a new DNA sequence.
- The specificity of HEGAAs is dependent on two things (1) the range of species the genetically modified virus can infect AND (2) the presence of the suitable DNA sequences in the plant chromosomes of cells that become infected.
- An example of an insect dispersed viral HEGAA which disrupts a specific plant gene is illustrated in this figure below
HEGAAs can be thought of as having two classes, depending on the biology of the viruses selected for development
- Somatic HEGAAs: all genetic alterations introduced into plant or animal chromosomes never impact seeds
- Germ-line HEGAAs: genetic alterations can impact seeds - it is probably not an understatement to say that it is hard to conceive of a type of synthetic virus of greater safety concern (see question below)
The difference between these two classes is analogous to the somatic gene editing of humans cells for medical reasons and germ line editing of eggs and sperm which impacts all subsequent generations
Prevention germ-line editing of seeds is an obvious safety criterion when selecting what viruses to develop HEGAAs from. Is there any evidence that avoiding of intentional or accidental germ-line editing was one of the requirements of the DARPA work plan?
- No
- Since the publication of the Science article in comments to the media DARPA project manger Blake Bextine did appear to make some constructive statements on the intentional or accidental genetic editing of crop seeds. Blake Bextine was quoted saying their “the program is not targeting the germline cells of plants” [1]. This is the kind of positive statement we might have expected more of in the DARPA response. However, even here the clarity of this statement is obscured by other program participants and other of Blake Bextine’s own quotes questioning any role of gene editing in the program [2], this is despite earlier clear statements to the contrary [3].Even, which, if any, of the 3 viral consortia's plans does not involve editing plant chromosomes remains unclarified by DARPA. Note that the acronym HEGAA partly derives from the term “genetic alteration agent” used in the DARPA work plan. See [references] on this link
- Note to edit the germline it is not necessary that HEGAA viruses be present in seeds (eg table A3 doi:10.1016/j.jtbi.2016.02.017), only that at some point during development they can infect and edit growing parts of plants that ultimately become flowering parts of plants meristems.
Is the Insect Allies program planing to release HEGAAs into the environment?
- No- all experiments within the current 4 year program (2017- 2021) are to be done in greenhouses. One press release mentions a BSL-3 facility.
What makes the DARPA Insect Allies program unique compared to all earlier GM virus programs?
- It is the very first program to propose or fund the development of HEGAA viruses. These have the capacity to perform genetic engineering in the environment (this might also include seeds if the target species is a plant)
- The insect based means of delivery mandated in the work plan for the genetically modified viruses is obviously unpredictable
- The motivations provided for earlier programs were clear and plausible
- The DARPA program is more easily weaponized than developed into an agricultural system sufficiently controllable to conceive of use in agriculture (this is nothing to do with the fact that DARPA is a military agency) - an earlier text of this clause was modified on the 6th of November
Why do we assert that it is almost always easier to develop a HEGAA biological weapon system than an agricultural one ?
- To conceive of a routine use in agriculture for HEGAAs it will be necessary to build in multiple mechanisms to control the spatial and taxonomic spread of the viruses. This will be complicated.
- When developing a biological weapon, most if not all ‘safeguards’ or ‘independent kill switches’ can be left out. Usefully the targeting of gene editing to your enemies crops is assured by the DNA sequence specificity of the 2 guide RNAs used by CRISPR (see Figure in Science paper, it is notable that the specificity of the guide sequences is one of the few elements that the insect allies program relies on which is in any way largely predictable )
- While to kill or sterilise a plant can likely be achieved by disrupting a single suitable gene, more complex traits will probably require inserting new genes or genes into plant chromosomes (examples repeatedly presented include resistance to drought, frost, flooding, salinity, herbicides, and plant disease). In most circumstances it is more than x1000 times more efficient to cause gene disruption than gene insertions (eg Mao, Z., Bozzella, M., Seluanov, A. & Gorbunova, V. Comparison of nonhomologous end joining and homologous recombination in human cells. DNA Repair (Amst.) 7, 1765–1771 (2008) ).
Has the Insect Allies program already had a biosecurity impact?
- Very difficult for us to say - but there this submission from a very distant branch of the US government to a meeting of the Biological Weapons Convention on August 16 2018 which is suggestive:-
- “Large-scale BW threats: We are concerned that some countries’ past biological weapons programs may continue to exist and new forms of biological weapons may be created through misuse of advances in science and technology. While the threat of deliberately caused disease outbreaks among people has received greatest attention, the United States is also concerned about potential attacks on crops and livestock.” Goto page 2 cited in Science paper as 14. United States of America, Strengthening of the BWC,Meeting of the States Parties to the Convention on the Prohibition of the Development, Production and Stockpiling of Bacteriological (Biological) and Toxin Weapons and on Their Destruction, Geneva (2018); http://undocs.org/en/BWC/MSP/2018/MX.5/WP.3.)
Have genetically modified viruses that have a capacity to edit chromosomes ever been developed?
- Yes. The following 2015 paper describes a genetically modified virus which when injected into the tails of laboratory mice can knock-out a specific gene in cells it infects. In this laboratory confined experiment after a week >40% of the chromosomes in liver cells had been irreversibly disrupted.
Ran, F. A.; Cong, L.; Yan, W. X.; Scott, D. A.; Gootenberg, J. S.; Kriz, A. J.; Zetsche, B.; Shalem, O.; Wu, X.; Makarova, K. S.; Koonin, E. V.; Sharp, P. A.; Zhang, F. In vivo genome editing using Staphylococcus aureus Cas9. Nature 2015, 520, 186–191, doi:10.1038/nature14299.
Are there natural plant viruses that have a capacity to edit plant chromosomes?
- No. Unlike animals plants do not have retroviruses that can alter their chromosomes.
Have genetically modified plant viruses that have a capacity to edit chromosomes in plant genomes ever been developed?
- NOT really. There are laboratory based protocols that use viruses to gene edit plant chromosomes but these currently require the infiltration and a bacterium to be effective (it is not the spraying of a virus onto plants). In a laboratory setting this dependence is partly a safety feature providing little motivation to remove it. It does seam reasonable that with sufficient effort it would be possible to generate viruses which carry all the requirements for a gene editing system. It is however conceivably possible to split parts into different released viruses (slide 15) .Note plant viruses can be selected to have broad host ranges. Viruses use in laboratory techniques for germl ine plant manipulation can in their wildtype state infect 100s of species across many Families , e.g. the papers cited in the Science article
- Z. Ali et al., Efficient Virus-Mediated Genome Editing in Plants Using the CRISPR/Cas9 System. Mol. Plant. 8, 1288-1291 (2015) .uses TRV= 400 species of plants from 50 families are susceptible to infection (nematode vector)
- K. Musiychuk et al., A launch vector for the production of vaccine antigens in plants. Influenza Other Respir. Viruses. 1, 19-25 (2007). uses TMV= nine plant families, and at least 125 individual species (insect vector).
- Hu, Jiacheng, Shaoya Li, Zhaolei Li, Huiyuan Li, Weibin Song, Haiming Zhao, Jinsheng Lai, Lanqin Xia, Dawei Li, and Yongliang Zhang. A Barley Stripe Mosaic Virus-Based Guide RNA Delivery System for Targeted Mutagenesis in Wheat and Maize. Molecular Plant Pathology 0, no. 0. Accessed July 23, 2019. https://doi.org/10.1111/mpp.12849.
What institutions are reported to be DARPA insect Allies contractors
This information all comes from press releases and as such my be neither reliable or up to date.
VIPER- Viruses and Insects as Plant Enhancement Resources (announced 27-JUL-2017[1])
Boyce Thompson Institute
UC Davis
Iowa State University
University of Minnesota
The final phase of the testing will occur in tightly controlled greenhouses at the University of California, Davis.
Up to $10.3 million
Target crop: maize
Insects mentioned: aphids, and leafhoppers
Ohio Consortium (announced December 20, 2017 [2] and October 17, 2017[3])
Ohio State
USDA Agricultural Research Service (ARS)
North Carolina State University
Oklahoma State University
“The study will be conducted at an ARS biosecurity level 3 facility at Ft. Detrick in Frederick, Maryland, with all testing done inside.“
up to $10 million.
Target crop: maize
Insects mentioned: leafhoppers or aphids
Penn State consortium - (announced November 20, 2017[4])
Penn State
University of Florida
University of Texas
Pacific Northwest National Laboratory
North Carolina State University
Kansas State University
Up to $7million
Target crop: Tomato
Insects mentioned: whitefly
[1] ‘BTI Receives DARPA “Insect Allies” Award’, EurekAlert!, 2017 <https://www.eurekalert.org/pub_releases/2017-07/bti-brd072717.php> [accessed 7 October 2017].
[2] ‘Ohio State Scientists Make Plant Virus System “Turn on Its Head” with Insect Research’, 2017 <https://www.thelantern.com/2017/12/ohio-state-scientists-to-make-plant-virus-system-turn-on-its-head-with-insect-research/> [accessed 4 February 2018].
[3] ‘Insect Allies: How the Enemies of Corn May Someday Save It’, 2017 <https://cals.ncsu.edu/news/insect-allies-how-the-enemies-of-corn-may-someday-save-it/> [accessed 2 February 2018].
[4] ‘Penn State Team Receives $7M Award to Enlist Insects as Allies for Food Security’, 2017 <http://news.psu.edu/story/495037/2017/11/20/research/penn-state-team-receives-7m-award-enlist-insects-allies-food> [accessed 2 February 2018].
There is a 4th consortium funded as part of the Insect Allies program but it does not involve genetically modified viruses, so we have opted not to consider it .
Can farmers currently spray viruses on their crops ? Why mandate insect-based dispersion at all?
- Our inclusion of a sentence of text about this topic in the Science article was not to advocate for the spraying of HEGAAs, it was to highlight the implausibility of the sole motivation so far presented by DARPA for mandating insect dispersion. None of the authors are farmers or knowledgeable about spraying
- Yes GM viruses can be sprayed onto plants to infect them in fields. In Kentucky field trials , “Virions were mixed with an abrasive and spray inoculated on either greenhouse-grown or field-grown plants” see review in Pogue, G. P.; Vojdani, F.; Palmer, K. E.; Hiatt, E.; Hume, S.; Phelps, J.; Long, L.; Bohorova, N.; Kim, D.; Pauly, M.; Velasco, J.; Whaley, K.; Zeitlin, L.; Garger, S. J.; White, E.; Bai, Y.; Haydon, H.; Bratcher, B. Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems. Plant Biotechnology Journal 2010, 8, 638–654, doi:10.1111/j.1467-7652.2009.00495.x.
- Though for a counter view “To enter plants, viruses have to get through the tough cell wall,” he says. “The wounding of the plant cell is in the vast majority of cases achieved by the vector entering the plant cell to feed, and in [most] cases this is by insect vectors—typically aphids, whiteflies, or leafhoppers.” see The Scientist
The history of non-HEGAA genetically modified virus developments
This is a link to a .pdf of recent talk by one of the authors, about their history and what makes viral solutions so outwardly attractive— note that this is a talk and information was intended to be provided verbally so please check before relying on any interpretation of the slides—
The GM citrus virus techniques mentioned is currently experimentally planted at a single 400 acre site in Florida there is a pending application to expand that to 67 sites and 513500 acres (USDA 17-044-101r)