Neil Stoker 07 September 2016 9976 words Submitted in partial fulfilment of the requirements for an MSc in Science Communication at Imperial College London
How new technology affects the research process: The impact of CRISPR
Contents Abstract ................................................................................................................................................... 2 1. Introduction ........................................................................................................................................ 2 2. Is CRISPR as a transformative technology? ......................................................................................... 6 2. 1 Introduction ................................................................................................................................. 6 2.2 The need for gene editing ............................................................................................................. 7 2.3 Uptake of CRISPR by scientists .................................................................................................... 12 2.4 Of mice and other model organisms .......................................................................................... 13 2.5 Beyond the supermodels ............................................................................................................ 19 2.6 CRISPR and other gene editing methods .................................................................................... 22 2.7 Unpredictable consequences and new imaginings..................................................................... 26 2.8 How does CRISPR compare to other transformative methods? ................................................. 28 2.9 Changes in the social world of the scientist................................................................................ 29 2.10 Conclusions ............................................................................................................................... 30 3. Is CRISPR democratising genetics research? ..................................................................................... 31 3.1 Introduction ................................................................................................................................ 31 3.2 Where are people using CRISPR? ................................................................................................ 34 3.3 Who is using CRISPR? .................................................................................................................. 38 3.4 Conclusions ................................................................................................................................. 42 4. Conclusions ....................................................................................................................................... 42 5. Methodology ..................................................................................................................................... 44 6. Appendices ........................................................................................................................................ 46 1: Interview transcripts ..................................................................................................................... 47 2: Survey results ................................................................................................................................ 47 3: Ethics form .................................................................................................................................... 47 7. Bibliography ...................................................................................................................................... 47
1
Abstract Gene editing of mammalian cells and embryos using CRISPR-Cas9 was first achieved in 2013, and the first editing of non-viable human embryos was reported two years later with wide media coverage. While controversial in this respect, the technology has been widely and rapidly adopted for use in many other organisms. In this dissertation I explore this technological revolution from the inside of the world of biology, using objective bibliometric analyses in conjunction with interviews with the researchers involved. Though still in its infancy, I demonstrate that the technology is being used much more frequently and much more widely than the previous existing gene editing techniques, and explore the reasons for this. CRISPR is experienced by researchers as transformative because of its combined flexibility, reliability, ease of use, and low cost, comparable to the invention of the polymerase chain reaction in 1985. I argue that simple gene editing completes a genetic toolbox that researchers require for genetic studies, and that this is a significant event that deserves study both in its own right, and as an example of an incipient technological revolution. There is also evidence that the simplicity of CRISPR-Cas9 technology is democratising gene editing in comparison to earlier methodologies, in the sense that it is being used more widely.
1. Introduction “China shocks world by genetically engineering human embryos � was one of many newspaper headlines on 23rd April 2015 (Knapton, 2015). A research paper had been published in which precise genetic changes had been created in human embryos, and there was a furore about human embryos being altered (Liang et al., 2015). Six months later, images of extra-muscly beagles announced that the first gene edited dogs had been born (Dvorsky, 2015), to a general reaction of passing interest rather than alarm.
2
I was struck by both of these events, my first real moments of awareness of gene editing, and my reactions were different to those I saw of others1. I was less concerned about the human experiment: it used non-viable embryos, as do many research laboratories, and I felt this hype obscured the far more relevant and exciting prospects of therapeutic somatic cell editing. I was more concerned about the second, because I felt that our genetic world had changed fundamentally, with what might now be done to any animal and plant we cared to think of. I was also astonished that making targeted genetic changes had improved a million-fold from what I had known to be possible. How had that slipped under my radar?
CRISPR was the name given to the technology, and soon was everywhere, both in the scientific journals, and the wider media.2 The Liang human embryo paper was a trigger for the world’s attention, but I realised that gene editing methods had been working for many years before that, gathering momentum. It became apparent that we were within the early stages of a major technological revolution which would impact society in many ways.
As an ex-scientist, I wanted to explore how this was being experienced by the scientists themselves. They are the first to hear of these developments, to think about them, and to have their mental, social and physical worlds changed by them. There are incremental advances all the time, but how are they affected by major technological possibilities?
1
Genome editing and gene editing are synonymous, and here I will use ‘gene editing’ throughout, as it is now more common. 2 "Clustered Regularly Interspaced Short Palindromic Repeats", which together with CRISPR-associated (Cas) proteins make up the system, but "CRISPR" is how the whole system tends to be referred to.
3
Science as social That science is a social as well as an intellectual process has been argued since the mid-20th century, with the work for example of Robert Merton, who in 1942 described the normative ideals within which science appeared to operate (Merton, 1973).
The results of science, though, the facts produced, appeared sacrosanct - objective and authoritative - until the 1960s, when Thomas Kuhn’s seminal work (Kuhn, 1962b), led the way for a new fields of sociological research, the sociology of scientific knowledge, and science and technology studies.
‘Laboratory studies’ are anthropological approaches pioneered by Latour and Woolgar (1979), to study the social world of the scientist. They and Knorr-Cetina (1999) described molecular biology research from within the laboratory, how ‘facts’ are constructed, and how they vary in certainty. It was shown that the journey from idea to experiment to interpretation of data to general acceptance, is a complicated one that occurs within a social world.
It is therefore reasonable to study the social world of science. Indeed, it can be argued that the role of science is so important today, involving so many people, such large resources, and with such farreaching consequences - it is invested with such power - that it would be delinquent not to study it from a social perspective in detail.
Technology in biological research If we ask what has driven the extraordinary development of science over the past 400 years, there are many contributory factors: the scientific method, new social attitudes, the development of systematic collaboration (Zilsel, 1945), intellectual and conceptual advances, the investment of time, money and people, or collective memory through publication.
4
These may all be important, but I’m interested here in the technology which, I believe is high on the list. Even with publication, perhaps it was the technological advance of printing over writing (Eisenstein, 2002) that made science as we know it possible, and allowed an exponential increase in knowledge. Furthermore, development of technology is itself an exponential self-replicating process, in that each development breeds multiple new ideas both about what it could be used for, and about new technologies we might aspire to. As Sydney Brenner said, “Progress in science depends on new techniques, new ideas, and new discoveries, probably in that order” (Robertson, 1980). Examples of the bidirectional interplay of technology and research in physical and biological sciences are discussed respectively by Townes (1983) and Fields (2001), while Traweek (1988 p49) reflects that in the world of high energy physics, "inventing machines is part of discovering nature".
When reading Thomas Kuhn’s descriptions of ‘normal science’ (Kuhn, 1962a), one of my overriding impressions was of my own experience of scientists who will often know that what they are doing is imperfect and will soon become trivial or redundant, but they are doing the best they can with the tools they have. They are not intellectually unaware, which is how ‘normal science’ is sometimes portrayed (Popper, 1970), but technically hamstrung, doing what is possible while waiting for different tools that will either give the answer they are looking for, or will make new questions possible.
Even intellectually, we sit within paradigms not necessarily because we believe them, but because they allow some action; to think beyond these is mere speculation. So for me, discontinuity in progress is often caused by new technologies that act as bridges into worlds to which we had no access before. They might be bridges that are completely new, or ones that offer much greater access than previously existed, available not just to pioneers, specialists or privileged few. Technology changes what we can think, and what we can usefully think. I would argue that the current excitement over gene editing represents one of these moments. 5
Aims Here I want to investigate the social world of the scientist from a different perspective from that of how knowledge is made. This study will also differ from earlier ones cited, in that I am closer to the science; my background is in genetics, whereas Latour, Woolgar and Knorr-Cetina came from philosophy, sociology and cultural anthropology backgrounds. I want to take advantage of being within a technological revolution, and use the example of gene editing to explore how technological advance is experienced by the biologists themselves, and to explore the nature of this particular technology. I was keen to be able to present the voice of the researcher, normally heard only through publishing or promotion of results, talking about the everyday realities of research as they experience it. This is therefore more of an emic rather than an etic approach (Harris, 1976).
I will do this partly through interviewing scientists who are actively using CRISPR, and explore whether:
1. They experience CRISPR as transformative, and if so how? (Section 2)
2. CRISPR is democratising research (Section 3)
2. Is CRISPR as a transformative technology? 2. 1 Introduction Is CRISPR transformative? Some of the language used by the scientists here (“Game changer”, “Blowing my mind”) certainly suggests so.
For a technology to be transformative, it must first solve a need that is important and relevant to people involved (section 2.2).
6

Secondly, it needs to actually be taken up, that is to be transformative as well as having the potential. I discuss bibliometric data (section 2.3), its use with model organisms (section 2.4), and its wider use (section 2.5).

Thirdly, it will do its job, and fit into the research process, better in some way than alternative methods (section 2.6).

Fourthly, it might be transformative through unpredictable consequences (section 2.7)
All these are interrelated, so cannot be completely separated. I have used bibliometric analyses, interviews, and a survey to inform my text.
2.2 The need for gene editing The discipline of genetics Genetics is a discipline that describes the genetic content of an organism, and to understand its functions. Although disciplines overlap, genetics essentially looks at information content and flow. There has also always been a parallel applied strand that seeks to improve farm animals and crops, and relieve genetic disorders in humans. Figure 2.1 shows key dates in conceptual understanding, achievements, and technical breakthroughs relevant to CRISPR.
7
Figure 2.1: Key dates in the development of genetics research. Items in red italics: Development of critical methods for genetic analysis. Gene editing dates are first use in vivo.
‘Gene editing’ is really just a fancy new term for making mutants, albeit with enhanced intention and accuracy. Mutants - or genetic variants - have always been at the core of genetics. The only practical way to understand such complex systems is to alter them while keeping the system intact. So the function of components is analysed through difference, using normal genetic variation, or deliberately creating mutants. In this way, a the genetic information in a complex organism can be mapped, and functions assigned.
Moving from random to targeted mutagenesis Until the 1970s, mutations were obtained by treating bacteria and fruit flies with mutagenic compounds, and screening (hundreds of) thousands of colonies or flies for ones that are different (St
8
Johnston, 2002, Shuman and Silhavy, 2003). Then with recombinant DNA and sequencing technologies, the situation changed, with the gene identified first, and a subsequent search for its function (reverse genetics). Targeted mutagenesis was first achieved in mice in 1987 (Thomas and Capecchi, 1987). The process, called gene targeting, was through a laborious process (Figure 2.4).
Gene editing: the last tool in the box? By 2000, most requirements for genetic analysis were in place, except for - with most organisms efficient targeted mutagenesis, and gene editing completes the genetic toolbox. It is what researchers have been working towards since gene cloning appeared in 1973. As Figure 2.2 shows, techniques in this toolbox are not independent, but all rely on each other.
Figure 2.2: The Genetic Toolbox: key technologies for genetic research. Arrows indicate that one method is required for, or informs, another. Gene editing dates are first use in vivo. NGS: Nextgeneration sequencing; PCR: polymerase chain reaction 9
All methods of targeted mutagenesis require a way of directing a construct to a specific DNA sequence, and also the existence of active DNA repair or recombination systems within the cell to enact the change.3
Classical gene targeting was extremely inefficient (~one successful event in a million). Gene editing is a different approach, and the game change is that its efficiency is no longer 10-6, but can approach 100%.
Gene editing currently comes in three different flavours: zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and CRISPR (Table 2.1). 4 Details of how they work are reviewed by Kim and Kim (2014)
Gene targeting uses homologous recombination (HR), a normal cell process, which is slow and inefficient without initial cleavage of the DNA.
Gene editing is based around first cutting the genome at the site where change is desired. Once that is done this break can be mended either through a DNA repair pathway (non-homologous end joining; NHEJ), which can result in mutation, or HR using DNA introduced at the same time. The main difference in techniques revolves around how the sequence of interest is targeted so that the initial cuts are made:
ZFNs:

Zinc finger (ZF) proteins are eukaryotic gene regulators that bind to specific sequences through their ZF domains, and switch genes on and off.

3 4
A ZFN is an artificial protein where the ZF part of one protein is fused to a nuclease domain.
Transient methods for studying gene function such as RNAi not included for space and clarity. A fourth, meganucleases, is so limited in sequences it can target, that I will not discuss it here.
10
Each ‘finger’ binds three bases, so by chaining together ZFs with different specificities, any DNA sequence can be targeted.
These constructs have to made individually, and is a difficult process.
TALENs:
These are also artificial sequence-targeted nucleases.
Instead of ZF domains, the binding is based on proteins (Transcription Activator-Like Effectors; TALEs), made by bacteria that infect plants.
A TALEN is a TALE domain that is fused to a nuclease domain.
The major difference with ZFNs is that TALE domains bind single bases, not triplets, so you only have to deal with four types of subunit; by linking these in a chain, any DNA sequence specificity can be obtained
These constructs have to be custom-made individually, a moderately difficult process
CRISPR:
This is based on a bacterial antivirus system
A short hybrid RNA (guide) molecule is used where half binds to a cleaving protein (Cas9), and the other half binds to the desired cleavage site in the genome
Specificity is purely in the RNA sequence, which can be ordered online
For all, once made, the actual targeting of the protein to the DNA of interest is rapid and efficient Table 2.1: Gene targeting and gene editing methods compared
Conclusion I started by saying that a transformative method should solve a need. The ability to make genetic changes at will is a core requirement of genetic research. This had been difficult or impossible in most organisms, and gene editing enables it in a more efficient and universal way. All gene editing methods satisfy this requirement. 11
2.3 Uptake of CRISPR by scientists If they all solve a need, is CRISPR transformative in that it is being used more that the other methods? The most obvious measure of the usage of a technique is through bibliometric analysis. I therefore compared the number of primary research papers using CRISPR compared to ZFNs and TALENs (Figure 2.3).
A
B
Figure 2.3: Primary research publications using different gene editing technologies by year. A: Global publications; B: Publications with UK authorship. 2016 data are extrapolations from August, when the data were collected.
CRISPR gene editing papers are much higher in number, and increasing exponentially. There is a large difference between CRISPR and other methods, and the CRISPR papers are rising exponentially. The other methods, invented earlier, were never highly cited; the number of TALEN papers rose to 2014, but plateaued, presumably due to the uptake of CRISPR.
Conclusion I conclude that CRISPR is transformative in that is being actively and successfully used significantly more than competing technologies, which were developed earlier. 12
2.4 Of mice and other model organisms Model organisms help focus research It makes sense for scientists to focus on particular ‘model’ organisms when researching fundamental mechanisms , so that results can be shared, and toolkits developed. If I want to see the gold standard for research methodologies, then this is the obvious first place to look. Table 2.2 shows a list of some of the most widely used models – what you might call the ‘supermodels’ - with very large research communities.
Organism
Model for
Mutagenesis methods before gene editing
CRISPR works?
Mouse
Higher mammals
Chemical Random insertion Classical gene targeting
yes
Arabidopsis thaliana (Thale cress)
Flowering plants
Chemical Radiation Random insertion Classical gene targeting
yes
Drosophila Insects, higher eukaryotes melanogaster (Fruit fly)
Radiation Random insertion Classical gene targeting
yes
Zebrafish
Fish, vertebrates
Chemical Random insertion
yes
C. elegans
Nematodes, neuronal/ cellular development
Chemical
yes
13
S. cerevisiae (budding yeast)
Simple eukaryotes
Efficient methods available
yes
E. coli
Bacteria
Efficient methods available
yes
Table 2.2: Model species of organisms widely used in genetic research. The main mutagenesis methods before gene editing are listed; random methods are in normal type and targeted methods in italics. Transient methods such as RNAi are not included.
I will now describe how individual researchers working on different model organisms have experienced the impact of CRISPR on their work, through interviews or a survey.
The mouse I will focus first on how genetics is used in the most widely used mammalian experimental model, the mouse.
Vlad Seitan is a researcher at Kings College London, who was recently appointed on a fellowship to work on a protein complex that is important for transscriptional regulation. He described classical gene targeting (Fig 2.4) from his perspective. This starts with creating a long piece of DNA. He described an example that took several experienced people six months to produce:
One of the biggest challenges actually was to actually make the construct, because they needed to be very big […] In the lab where I did my PhD …there was the postdoc and the postdoc left, and there was a couple of other postdocs that came and worked on the same project, to PCR and ligate together this mammoth 10kb construct, and then cloning it wasn't very easy either, then sequencing the whole thing, and I think that took about 6 months. 14
Once you have this DNA construct, you introduce it into embryonic stem cells:
Then you put this large construct into ES cells, and then, depending on your luck and the gene that you're targeting - whether it's expressed in ES cells or not you'd have to screen anywhere between 100 and 5-600 colonies to find maybe one that would be recombinant.
A cell carrying a mutation is injected into a blastocyst, and mice carrying your mutation are born.
Then if you want to study the function of that gene, to knock it out completely, you have to cross heterozygous mice to get a homozygous generation. So that's another few more months. Even assuming everything works 100%, you're looking at about a year minimum.
This is a difficult and uncertain process that takes at least a year, with no guarantee of success. Despite this, a staggering 25,000 mutant mouse strains have been generated in this way.(Skarnes, 2015)
I asked Vlad how CRISPR has changed things:
With the development of CRISPR technology, it makes things a lot easier. I think it has so many advantages over the old classical gene targeting approaches, if your requirements are to just get a straight knockout of a particular gene [...] With CRISPR you just order your oligos, you clone it into these plasmids, readily available. [...] You can just inject these constructs into single cell mouse embryos, and apparently you get an efficiency as high as 70%. [...] and then in the first 15
generation you might have a mouse that already has the mutation that you need. So it's a huge huge time saving.
Already, 18 months ago, astonishing efficiencies were being reported (reviewed by Singh et al (2015)). These not only produce knockouts, but very specific intentional changes by adding an extra oligonucleotide that guides the repair at the DNA break through HR (Wang et al., 2013). Even more remarkable was the demonstration in the same paper that several genes could be altered at once, and Singh remarks about her own experience:
These phenotypic results were obtained only 6 weeks after embryo injections were performed, in contrast to years it would have taken to generate and breed triple homozygotes from individually targeted ES cells.(Singh et al., 2015)
I posted a survey on a forum, and respondents supported this:
(1) Previously my lab generated two knockout mice over a span of 15 years. In less than 3 years, we have generated 14 mouse models including knockouts and knockins. (2) It's a complete game changer. Most obvious example is mice- we did two rounds of microinjection, got 35 liveborn and 29 had biallelic mutations in our gene of interest, four more were hets. This would have been unimaginable preCRISPR even for complete knockouts (and they would have meant months of ES cell work). (3) We have successfully made more than a dozen mouse lines and only one mouse model did not work. (4) Works very consistently (Mouse mammary cell line) 16
This is an extraordinary change. Every aspect of this is faster, more reliable, and takes less expertise (Skarnes, 2015).
Figure 2.4: Comparison for making generic mutant mouse using classical gene targeting and CRISPR gene editing. Mouse image: Creative Commons5
5
https://commons.wikimedia.org/wiki/File:Vector_diagram_of_laboratory_mouse_(black_and_white).svg
17
C. elegans Other organisms have different challenges. C. elegans, a tiny nematode with a generation time of 3 days, has become a model system for understanding fundamentals of cell differentiation.
Despite this, there was no way of creating targeted mutants. Instead, The chemical mutagenesis is used, where worms are fed a mutagen, causing random genetic changes. Individual worms with interesting changes are then picked, and the genetic change determined. Over the years a large collection of mutants has been generated by the C. elegans research community
I spoke to Michalis Barkoulas from Imperial College,:
What is particularly interesting with CRISPRs is that it's extremely easy to produce them. The way we get transgenics is by injecting PCR products into the gonad of the animals; we don't even have to clone it. If today we had the primers - these oligos to amplify and create the single guide RNAs, we would have the construct ready by the end of the day; [...] within a week we'll have a couple of generations already of this transgenic, so let's say, within 2-3 weeks we can study the phenotypic consequences in a stable mutant that we have obtained.
With C. elegans, creating a targeted mutant, previously impossible, now takes days. So CRISPR is again transformative within this community.
Arabidopsis Arabidopsis is a fast growing brassica, and the main model flowering plant. Existing mutagenic techniques either random mutagenesis and classical gene targeting. I interviewed Jose GutierrezMarcos from the University of Warwick. 18
[With both other methods] we have to screen a large population to be able to find the mutant in the gene that we're interested in. CRISPR gets around all that, allows us to target a region of the genome to create a mutation, and for us this is incredibly powerful,.
He said that with CRISPR they could have a mutant in 6 months, and they are getting better :
[The first mutant was] a major project, but we have now developed a technique that it makes super-efficient . Because of this project we had to develop a new strategy to do CRISPR in Arabidopsis. It's very very easy.
Zebrafish I also heard from a researcher working on zebrafish, who said:
[It works] every time. It targets the gene of interest and is cheap and easy. Knocking out a single gene in Zebrafish has not been easily possible before.
Conclusions
I got the impression, that in all the model systems described above, each worked on by large communities of scientists, a real block had been removed. Something that was a major effort had become fast and routine. It seemed as radical as comparing travel by sailing ship to the aeroplane.
2.5 Beyond the supermodels CRISPR has significantly improved options for genetic research in these ‘supermodel’ systems. However, most organisms, many of them immensely important, are not model systems. Perhaps 19
this is where CRISPR will have its greatest impact? Peter Sarkie, a C. elegans researcher from Imperial College thinks so:
We work on other nematodes. I think this is where it's really going to make a big big difference, because in model organisms often the toolkit has already been pretty well designed, there are other ways of doing things. But in a lot of nonmodel organisms, CRISPR may be the only way, and it may allow non-model organisms to start to be experimented and manipulated in a way that just has not occurred before, and I think that for us is the most exciting aspect of the development of that technology.
A list of examples of non-model species for which CRISPR has been successfully used is shown in Table 2.3.
Primates: ●
Human cells, embryos
●
Macaque
Mammals:
Plants: ●
Pig
●
Wheat
●
Dog
●
Rice
●
Rabbit
●
Maize
●
Goat
●
Millet
●
Cattle
●
Soybean
●
Sheep
●
Purple false brome
●
Ferret
●
Sweet orange
●
Liverwort
●
Salamander
Birds:
Amphibia: ●
Chicken
20
● Fish:
Xenopus
Invertebrates: ●
Atlantic killifish
●
Silkworm
●
Nile tilapia
●
Anopheles mosquito
●
Atlantic salmon
●
Aedes mosquito
●
Japanese rice fish
●
Butterflies
●
Lamprey
●
Water flea
●
Sea urchin
●
Sea squirt
●
Sea anemone
Fungi:
Filamentous fungi
Candida albicans
Protozoa:
Bacteria: ●
Plasmodium
●
Streptococcus
●
Toxoplasma
●
Lactobacillus
●
Cryptosporidium
●
Clostridium
●
Streptomyces
Table 2.3: Non-supermodel or economically important organisms in which CRISPR gene editing has been successfully used.
It is remarkable to have proof of principle in so many organisms in such a short time. But in fact there is little depth so far, and many of these and others had been edited using ZFNs or TALENs. There are also organisms that are still hard to edit: bacteria (which generally don’t have suitable repair systems) and birds, where early embryos within the egg can’t currently be modified directly.
Another reason in terms of purely basic science to look beyond supermodels, is that they mislead us. We have to extrapolate from these to similar organisms, and those assumptions are often wrong. I 21
encountered this repeatedly when working on mycobacteria, whose biology is often very different from E. coli, and Peter Sarkies found the same:
I think the next stage of biology is probably going to move away from the model organism towards looking in a less biased approach. People used to make broad statements like 'in nematodes, there is no DNA methylation' based on C. elegans, and that's not true because in more ancestral nematodes it exists, and this is the kind of thing I think where the future of biology lies.
Conclusions
This use in non-model organisms - the breadth of application - will, in my view, become the most important aspect of CRISPR technology, and also one that is likely to raise major questions about public concern and need for engagement. At the moment, flags have been planted in the sand, but this has not really taken off yet. It is too soon to judge properly.
2.6 CRISPR and other gene editing methods The next question is whether CRISPR is transformative in comparison to ZFNs and TALENs. Table 2.4 shows my perception of the difficulty of using different methods, and CRISPR appears the easiest method:
22
Make construct
Introduce
carrying change
construct into
% Success
organism / cell
ZFNs
Hard or
Easy
Medium
expensive
TALENs
Medium
Easy
Medium
CRISPR
Easy
Easy
High
Table 2.4: Comparing relative difficulty of gene editing methods
ZFNs were invented 20 years ago in 1996, shown to work inside cells in 2001 (reviewed by Carroll (2011)), and I wanted to know why they hadn’t taken off as CRISPR has. ZFN expert Marcus Noyes from NYU University explained that because of the commercial potential, a company, Sangamo Biosciences, was formed in 1995 resulting in a monopoly (Scott, 2005):
Many of the main guys were actually in Carl Pabo's lab, and they ended up at Sangamo in California, and [...] they bought all of the intellectual property that they could get their hands on, in order to start this company, and for roughly 20 years they really had a stranglehold on the market, because if you wanted to do any therapeutic application of nuclease work, you really had to work with them.
23
The stranglehold included at first Sangamo developing custom ZFNs only in-house, only working with selected academic labs, and charging $25,000. That is affordable as an item in a $500,000 research grant, and this was not a simple procedure, so not unreasonable:
As a principal investigator, if you have no experience with zinc fingers, it might take you 2 years to master some technology to produce a set of zinc fingers to bind to their target, and then they might not be all that great to begin with.
But that price bracket is not exactly encouraging risk and creativity within the research community, and Sangamo were also being selective about who they worked with. The arrival of TALENs changed things:
[Sangamo] licensed a lot of their technology to Sigma Aldrich, and there I believe they were selling zinc finger nucleases for $5000 [...] dropping their prices to compete with TALENs.
Michalis Barkoulas described the impact of TALENs:
TALENs was a very huge discovery as well; [so when they arose,] everybody was very excited about that. Zinc finger nucleases were always tricky because there was monopoly, there was industry involved that you had to pay loads of money. So then TALENs came and were much better in the sense that they were molecular biology products you produce in the lab which were much more complicated than CRISPRs but still global, so it would take a couple of days to produce them
24
Gene editing and CRISPR: What is different? Proof of principle of the CRISPR approach to genetic modification was published in 2012, multiple uses in vivo in 2013, and reports of its use have risen exponentially since then, easily outstripping papers using ZFNs or TALENs (Doudna and Charpentier, 2014). What is different about CRISPR genome editing for researchers?
Factors that seem important differences to me include:
Off the shelf reagents. With both ZFNs and TALENs, there is significant investment and expertise needed before you start in order to create the targeting protein. With CRISPR, all reagents can be ordered commercially, and cell biology groups for example can carry out experiments without the need for someone who can clone genes. Indeed individuals can set up experiments without cloning expertise.
Cost. The cost of CRISPR reagents is much lower. A reusable plasmid from Addgene costs $65 + shipping, and oligonucleotides cost ~£5 each. 6,7
Reliability. The experiments are more likely to work. There is less to go wrong, alternative versions can be easily substituted, and controls are easier to construct. Michalis Barkoulas commented on how CRISPR compared to TALENs:
The problem though was that the efficiency of TALENs was not as good as CRISPRs, so you would have to produce a lot of TALEN constructs.
6
https://www.addgene.org/ordering/ https://www.thermofisher.com/uk/en/home/products-and-services/product-types/primers-oligosnucleotides/invitrogen-custom-dna-oligos/oligo-ordering-details/pricing-for-oligonucleotides.html 7
25
Low bar for trying. CRISPR can be tried without investing significant resources, or before applying for funding. Multiple versions can be tested in parallel, allowing them to proceed with those that work most easily.
Low level of expertise needed. As Marcus Noyes told me:
The simplicity of it has opened it up to people with a huge range of expertise, and it works just as well for someone who just read their first Cas9 paper this morning, as it works for somebody like me, who has been studying this nuclease field for the last 15 years.
2.7 Unpredictable consequences and new imaginings A feature of transformative technologies is that they also allow people to think in ways that were not previously possible. Does CRISPR encourage innovation, and move beyond the uses that were originally thought of?
In some ways, this is just a general sense of new opportunities that are still hard to articulate. When asked if CRISPR was changing the way they thought, comments I received included:
1. Yes, definitely but it's hard to explain in how many ways this is true. It's a complete game changer. 2. Yes, but difficult to say how. Blowing my mind at the moment! 3. [There’s a] feeling of horizons opening up
Two specific examples were mentioned:8
8
Gene drives would be another example, but was not commented on
26
Multiple changes With classical gene targeting, only one gene could be targeted at once. With CRISPR, the efficiency is such that multiple identical sequences can be targeted at once. A researcher studying mouse epithelial cells told me:
It is helping nail down nagging questions that couldn't be addressed before, or were too hard/expensive to contemplate. EG: if I want to make a triple KO, I just do it, inconceivable preCRISPR
Genome-wide screens Another unprecedented development is genome-wide screens (Shalem et al., 2015). Instead of using one guide RNA, you can use 50,000. Marcus Noyes commented:
I think honestly, the nuclease technology for making modifications in a genome I think is the least exciting part about Cas9. I think the most exciting part is the screens that it enables us to now do, that we can build these huge libraries of mRNA, that maybe they target every possible splice junction, or promoter, or open reading frame, whatever you want your library to target, and then you screen through millions of cells to find ones that have some different behaviour, [...] that is fascinating , that allows us to research on a scale that we could never do before, and zinc fingers wouldn't have enabled that technology. The screens are radically different..
27
2.8 How does CRISPR compare to other transformative methods? If CRISPR is a genuinely transformative technology, how does it compare to other such methods? I asked people in person and through the survey, with an open question, but prompting them with PCR and NGS, because I did want to get a reaction to these.
Responses from senior researchers, who I felt would have more perspective were very clear that PCR was the technology this felt akin to.
(1) I was an enthusiastic early adopter of PCR, one of the first to publish using it for human genetics in the UK. Exactly the same feeling of horizons opening up. (2) This is the most revolutionary thing I have seen in 30 years even eclipsing PCR, iPS cells and so on, It may be the single most important innovation in the last 100 years or more! (3) It seems to me to be as game-changing as PCR (4) Indeed this can be considered as the last missing tech for genome manipulation, pcr was great, NGS IS GREAT and crispr is super great :-) genome editing affordable (5) This is the best thing since PCR
This is not a random sample, but it fits with my own experience. The PCR comparison is relevant in many different ways, combining to increase accessibility.
1. Transformation: There is a real sense of “before and after”. Having a sequence used to give you real power over others in the research race. 2. Speed: The time frame has collapsed. A major project that could take years became a routine afternoon’s work. 28
3. Cost: There is the same reduction in cost from a major budget line on a grant to small change. 4. Expertise: The level of expertise needed plummeted. What required great technical skill and implicit knowledge, could be achieved by undergraduates. 5. IP: IP issues have not stopped academic use 6. Specificity: With PCR, as with CRISPR, lack of specificity was the main problem encountered; it took many years for PCR to largely overcome it, and CRISPR is only just at the start of that process. Marcus Noyes said:
Being able to go from what appears to be very good specificity to perfect specificity is something that took the zinc finger field like 20 years to do
7. Community: The large community using the tools greatly helped individuals with problems, and drive improvements and innovations. 8. Innovation: There seems to be simplicity and flexibility of the components that allows innovation. 9. Interdisciplinarity: The low entry threshold allowed people from other disciplines, with other expertises, to incorporate DNA technology into what they do. 10. Perceptions: Both techniques have changed the ways that scientists think about their research, and what is possible
2.9 Changes in the social world of the scientist How is the social world of the researcher changed by CRISPR? A real picture of this would require more of an immersion in the field, and perhaps an ethnographic approach, than I could achieve here. So instead I mention anecdotal remarks on funding.
29
Getting funding occupies much of the life of a senior scientist. To convince a funding panel, the minimum is to have a good idea, where you are likely to achieve the first parts of the project. In genetics, obtaining a mutant is often the first step, so for this to be difficult presents a major problem for applicants. Having a reliable and quick method changes things, as Vlad Seitan confirmed:
That actually came up in my interview for my fellowship because I wanted to study this new gene. I told them that I had two different approaches. One, the classical gene targeting, because there was a construct made out there in one of these large programmes of gene targeting, and then, as a parallel approach I would use CRISPR. Someone on the panel said, oh you only have the construct, you don't have the ES cells yet to make the mice, and I said, no but I'm doing CRISPR, and he said that's OK, he seemed to be reassured that if I'm doing CRISPR then things will work.
The other way of doing this is to carry out pilot studies that can be fed into the grant application - to have a mutant in the bank - and this becomes feasible to do ‘on the side’ with CRISPR. It is also possible to be more adventurous; a survey respondent wrote:
It has allowed us to consider riskier projects, because generating the mutants is less of an investment.
2.10 Conclusions I have provided evidence and arguments that gene editing is transformative for scientists in terms of what they can now do, and why CRISPR has taken off in a way that the previous methods did not. It
30
is changing the way scientists think and what they can do, or are required to do. There is a strong parallel with the introduction of PCR.
3. Is CRISPR democratising genetics research? 3.1 Introduction The word 'democratising' is now used quite widely in relation to CRISPR in opinion pieces (Ledford, 2015, Lunau, 2016, Travis, 2015). Is this true, and what does it mean? Simplistically, it means that the technology is accessible to a wider group of people than other similar technologies. However, it also carries overtones about an equalising of power, with the implication that limitations of access to technology leads to inequality of power.
Scientific capital and the Matthew effect Pierre Bourdieu (1986) articulated the idea that power within society is created not only through money (economic capital), but also through social and cultural capital that can be converted into economic capital. These might respectively include for example, one’s social connections and one’s education. One’s life in society is experienced through the lens of this power (or lack of it).
This is a useful concept for discussing the world of research: what we might call research capital (Green and Rein, 2013), and how this affects power relationships and can cause inequalities.
What is important here is whether, in the context of a scientist, an institution, or a country, components of research capital interact with the others, leading to positive or negative reinforcement, and I suggest that they do. For a prestigious institution such as Oxford University, second in the World University rankings (Times Higher Education, 2016), the many factors that constitute research capital work in a virtuous network, all affirming and amplifying the others in a 31
complex way (Figure 3.1). In regions with low research capital such as sub-Saharan Africa, individuals, the institution, and the country are multiply and continuously disadvantaged. Just as with compound interest, depending on whether you are lender or debtor, you become exponentially richer or poorer in comparison to the other; inequality increases. This is what Robert Merton (1968) called ‘the Matthew Effect' in science: “For whosoever hath, to him shall be given…”9. Traweek (1988 p109) notes how closed the research world is, with scientists having clear ideas of the statuses of both institutions and countries. She also sees how important oral communication is, by definition limited to people one is in close contact with, thus reinforcing the network (Traweek, 1988 p120).
Figure 3.1: A virtuous network of research capital for institution and researcher. In a less privileged environment, it becomes a vicious network
9
New Testament, Matthew 13:12
32
Some inequality is useful: centres of excellence can move science forward, and support the rest of the research community. But all countries, regions, and institutions need to be able to carry out high quality research relevant to their needs, to benefit education and life in those places, and to have the power to set their own agendas. Technology can help reduce or increase inequality, depending on its accessibility.
CRISPR to the rescue? For a technology to be democratising, just as for it to be transformative (section 2.1) it must first solve a need that is important and relevant. I have argued above that this is true for CRISPR, as gene editing is a critical technique for basic and applied science in all living things, with significant potential in economic and health-related products, such as improving food crops and farm animal stock, and providing tools for treating disease.
The second requirement to be democratising, is that it is either new or replaces something that is more exclusive. I have discussed that CRISPR is less exclusive in terms of cost, ease of use, effectiveness, access to components and know-how, and intellectual property (IP) issues (Section 2.6). A professional scientist working on zebrafish in the US supported this:
I do think [CRISPR is democratising], and maybe we are an example of this. We do the process very cheaply in the lab using home-made components, inject mRNA for Cas9, with home-made guides, and screen for disruption of RE sites at the cas9 cut site. Any lab could do this if they have a PCR machine and some reagents.
Another commented:
33
I like the idea that people can do CRISPR in a broom closet if they know what they are doing. Yes, it makes this type of science more accessible.
CRISPR therefore should be democratising research. From the exponential rise of the use of CRISPR in published papers (Figure 2.3), it is self-evident that the technology is being used more widely than previous forms of gene editing in some manner. However, it is worth analysing in more detail.
In this section, I will therefore look at two aspects of the use of CRISPR that relate to widening of access and use: where CRISPR is being used, and a particular aspect of who is using it.
3.2 Where are people using CRISPR? Widening of access and use could be geographical: an expansion to different countries, or within a country to more diverse types of institutions with different levels of economic and scientific capital.
I therefore examined the locations of authors of CRISPR papers, comparing to ZFNs and TALENs. Kuzhabekova and Kuzma (2014) collected some related data, but did not include CRISPR.
The global situation The overall numbers of gene editing publications are shown in Fig. 3.2, together with the geographical locations of the authors’ institutions.
34
ZFNs (581/45/237)
TALENs (509/39/237)
CRISPR (3137/75/667)
Figure 3.2: Geographical distribution of gene editing papers. Cities which have produced a paper using a particular technique are marked. Figure shown are (papers / countries / cities).
Inspection showed these data to be imperfect, but in terms both of specificity of topic and authors' locations, but I felt were good enough as a first pass, and I examine the UK in more detail below. Taking my caveats, in its much shorter existence, CRISPR publications have authorships in 75 countries and 667 cities, far more than any of the competing technologies.
35
My main observations from this series of maps are: (1) that there are three super-regions in this field - North America, Europe and the Far East (China/Japan); (2) that in these super-regions, CRISPR has been used in many more centres than the other technologies; and (3) there appears to have been a greater take up of the technology in other parts of the world, including India, South America, and Africa.
I suspect that the general assumptions about the super-regions are correct (see UK discussion below), but the scattering of locations outside these regiouns need to be treated with caution. I reality-checked all publications from specific regions, and the CRISPR figures are inflated through non-gene editing work, but not equally (Figure 3.3). This is less misleading in India, with major authorship in 11 gene editing publications. In contrast, most papers from South America and Africa, focus on bacterial CRISPR sequences/basic CRISPR biology, rather than gene editing. The UK data show significant active gene editing research.
90 80
No. of papers
70 60 50 40 30 20 10 0 India Major authorship
Central / South America Minor authorship
Africa
UK
Not Gene Editing
Figure 3.3: Relevance to active gene editing of papers from selected regions. Major authorship: At least first or last author is from that region. ‘Not gene editing’: Mostly CRISPR biology papers.
36
Locations of authors of genuine gene editing papers in the UK are shown in Figure 3.5. With ZFNs and TALENs that the researchers come from high prestige cities - London, Oxford, Cambridge and Edinburgh - but the numbers are very small. With CRISPR, these cities are most involved again, with multiple publications, but there is a plethora of other locations involved, 28 in total.
ZFNs (15/3; 6)
TALENs (15/6; 9)
CRISPR (82/39; 28)
Figure 3.5: Locations of UK scientists publishing papers using different gene editing technologies. Bubbles show locations of city/town containing institutions, with size proportional to number from that city. Each paper is only used once in any city. Numbers in brackets indicate (number of papers with first or last authorship / number of papers with middle authorship only; number of cities).
In conclusion, CRISPR is being used in a much wider range of cities and institution than previous gene editing methods. Overall, I conclude that these data support the idea that CRISPR is indeed a more ‘democratic’ technology in this sense of the word, and likely to spread much more widely in the future.
37
3.3 Who is using CRISPR? It could also be about the people, and their place in the science world. If democratising, I would expect that the type of person using CRISPR is broad. This is something that is hard to study in a systematic or quantitative way, and I use anecdotal observations here to inform the discussion.
Undergraduates
This technology is already being used by undergraduates. Michalis Barkoulas commented:
CRISPRs is something undergraduates do in the lab routinely, doing PCR techniques you can easily create within a couple of hours a CRISPR construct that you can inject into C. elegans, and target a certain gene to modify and edit that particular locus.
These will be students doing projects in the laboratory, and you need techniques that are quick, simple, robust, flexible, cheap, and allow intellectual input without too much training. CRISPR allows that this, so that new and different projects can easily be set up and undertaken by each student.
Citizen scientists
The actual practice of science is a closed world in many ways. It happens within specialist facilities; it uses unfamiliar equipment and materials; it has its own language in which it publishes its results, often behind a paywall; and even scientists themselves are bemused outside their own specialty.
The public engagement movement, signalled in the UK by the Bodmer report (Bodmer, 1985), is partly led by the recognition by scientists that there needs to be greater communication with the 38
public. In some forms this has led to active involvement of non-professionals in the scientific process itself, often called Citizen Science (Davies et al., 2016). This is usually through a connection to projects driven by scientists (Zooniverse, 2016), though in some cases there is co-ownership with the public (Leydesdorff and Ward, 2005).
Biohacking or DIYBio (Seyfried et al., 2014) is rather different, firstly because it involves laboratory work, and secondly because the experiments are conceived of by the amateurs themselves. This movement in molecular biology is encouraged through the iGEM project for synthetic biology (iGEM, 2016), through competitions where professional, student and amateur scientists can compete on an equal basis.
In response to my survey posted on a CRISPR technical forum, I heard from biohacker who is using CRISPR.
Dan Wrister is a lawyer by day in Los Angeles, and on evenings and weekends 'plays' in an amateur laboratory (THELAB, 2016). He started with PCR because of its connection to his day job:
They were using it to identify criminal suspects, I was working criminal law, and I figured it would be good to know that.
So he got into the field from the desire to understand something important to his work, but closed to him as an expertise from a different world, that he was required to engage with. He did some PCR, and then he started learning some gene cloning techniques, and moved on to CRISPR:
39
When CRISPR came along, 'oh good, there's something that's brand new, that ground-breaking, and I can be up to speed almost the same as everyone else [...] it was nice to get in on the ground floor, and participate.
There was a buzz with its newness, and a sense of equality with scientists. His group did have a couple of experienced (PhD level) members who could help, but Dan was able to tap into a much wider community of practising scientists through online forums:
The way they've developed it, Feng Zhang, who's got the patent now, it's kind of open source, the development, and I think it's developed so rapidly because of the openness, I mean you can just join the Google group, and you'll learn a tremendous amount of information. There's several questions every day, and it's really helpful. I can write a question myself, and Feng or some of the leaders in the field, will answer it that same day.
Not only can Dan read real scientists' conversations, he can join in, and even get replies from CRISPR 'royalty' – the inventors. He is carrying out what he calls ‘simple’, but is actually a complex project using Arabidopsis:
My CRISPR project is a simple knockout of a plant gene called the 'leafy' gene, Lfy, and I'm just going to knock it out, and then I'll have visual confirmation if it works, because it will never flower
He has been to a major science meeting:
40
I went to the genome engineering 2.0 at MIT, they let me come as the only biohacker there , but it's like wow, there's George Church taking to us about our lab, and here's Feng Zhang, and they couldn't have been any nicer or more gracious.
Above all, it’s exciting:
It's just a different world, and it's fascinating . I think there's so much potential, more than anything else I think right now.
Dan also described meeting young people getting into biohacking at an iGEM event:
We met a 14 year old boy and a 12 year old boy, and it was a College and Graduate School iGEM, but they were absolutely brilliant, really, unbelievably love this stuff, and they live it. They know so much more than me or a lot of other people published getting their PhDs, it’s just amazing how much a 14 year old knows. He’s won the state science fair, and he’s building plasmids at home constantly, it’s amazing.
This doesn’t surprise me, as molecular biology is like being given the keys to the candy store, with catalogues full of thousands of molecular tools that you can use to solve practical puzzles. There are laws to uncover, and these are ‘real’ as opposed to being invented for a game; there is an element of creativity combined with precision. It’s like computer programming or music technology, and these people can just have fun, or they might become very successful and very rich. Either way, they can be involved, and without spaces like this, they are completely excluded from any practical involvement. 41
3.4 Conclusions I have shown that, compared to previous gene editing methods, CRISPR is being used more as a technique, in a wider spread of institutions both in the UK and throughout the world, and that its use has spread to undergraduates and amateurs.
That wider use could be described as a democratisation. I have argued that it is extremely important, and that there are ramifications that reach deep into the structures of societies. Gene editing is just beginning, and it will be interesting to follow how it spreads into different parts of science, and throughout the world.
4. Conclusions I have explored the importance of gene editing in general, and CRISPR in particular, to biological research. I have argued that gene editing is the last key component of the genetic toolbox, and that CRISPR is already transforming research into model organisms, and the signs are that it will be able to expand into the relatively uncharted territory of the rest of biology. Time has collapsed: What took months takes weeks, what took years takes months. What was impracticable can now be done, and completely new possibilities are emerging.
I have shown that CRISPR has taken off far more rapidly that ZFNs or TALENs, through its accessibility, and the lack of technical expertise needed. I have argued that this is democratising, and allowing the spread of a technology that can empower people, and potentially reduce inequality. It is even leaking out of the professional world into the amateur.
The similarity to PCR is the one most often drawn. If you look at the timeline of PCR’s history you can see that in 1989 (four years after its invention), it is still in its infancy (Figure 4.1). Similarly, CRISPR is
42
right at the start of its history. It may be that it is superseded by an even simpler method, but gene editing has come of age.
Figure 4.1: PCR citations by year. 2016 figure is a projection.
I have combined bibliometric data that shows objectively that a major shift is taking place, with an emic approach - reporting the comments of the scientists involved - in order to reveal how this shift appears from the inside of the research world. Much of work looking at the sociology of scientific research is etic, with complete separation is seen as a virtue. It allows ethnographic studies to be carried out where every aspect of the strangeness of laboratory life can be appreciated (e.g. Latour and Woolgar, 1979). However the issue of objectivity is complex (Hegelund, 2005), and I suggest there is also a place for observation by people, such as myself, who are cultural insiders. I am trying less to critique the existence and structure of the research world, and more to describe how it appears from the inside, and how change is experienced. Emic and etic approaches can be regarded as complementary, and lie on a spectrum rather than being dichotomous (Berry, 1999). As well as usefully broadening the perspectives from which research is documented, this may also encourage dialogue between sociologists and laboratory scientists.
43
5. Methodology Bibliometric data These were accessed PubMed in July and August 2016; my controls with which to compare CRISPR were the two preceding gene editing methods.
●
The main search terms were: ○
"zinc finger nuclease*" or "zinc-finger nuclease*" or zfn*
○
(TALEN* (not talent*)) or "TALE nuclease*" or "TAL effector
○
"CRISPR"
nuclease*")
●
UK-specific papers were found by adding: ○
●
Date of publication was limited through adding (e.g. for 2013): ○
●
AND ("United Kingdom"[ad] OR "UK"[ad] OR "U.K."[ad])
AND ("2013/01/01"[DP] : "2013/12/31"[DP])
Throughout, only papers since 2000 were counted.
The list of references was then inspected to keep only those that appeared to be primary research or methods papers on gene editing. Reviews, papers looking at basic CRISPR biology in bacterial systems, papers focusing overwhelmingly on a different gene editing technology, and irrelevant references were removed.
All references were inspected, unless there were more than 100 references within a year. In this case, 50 references were sampled at equal intervals throughout the reference set (e.g if 200 references, every 4th reference was inspected), and the figure obtained was used to calculate the likely total.
44
PCR: Searched at PubMed using ("polymerase chain reaction" or PCR not review), 05 August 2016, Total papers 648796.
Where 2016 data are graphed, the projected figure was calculated from the date the count was made.
Geographic data World map data were obtained at GoPubMed – a PubMed portal. Bubble data were obtained through the searches described above, and relevance and author locations manually ascertained, and plotted at MapsData; a high value distant city was included, to make different maps comparable.
Interviews People were selected initially using an internet search for CRISPR use at Imperial College (#1), and through a personal contact in the human genetics field (#2). These interviews were carried out in person, and allowed me get a feel for the area. I was interviewing #3 and #4, by phone for a different purpose, and asked them about their CRISPR use. #5 came from the survey below, and was interviewed as a very different type of user. Finally, I wanted a ZFN user perspective, so I posted a request on the CRISPR forum, and received a recommendation that led to #6.
1. Michalis Barkoulas (Imperial College London - C. elegans genetics) 2. Vlad Seitan (Kings College London - mouse genetics) 3. Jose Gutierrez-Marcos (University of Warwick - plant genetics) 4. Peter Sarkies (Imperial College London - C. elegans genetics) 5. Dan Wrister (Los Angeles - TheL4b biohack lab) 6. Marcus Noyes (NYU University - ZFN expert) 45
Survey A survey was produced in SurveyMonkey (see Appendix), and posted on the Google Group "Genome Engineering using CRISPR/Cas Systems" forum, which has 4676 members (24 July 2016).10 There was no attempt to get representative responses; the aim was merely to get extra viewpoints by asking a much wider pool of people, to find if I had missed anything significant, and to make contact with individuals whom I might interview.
Abbreviations Cas: CRISPR-associated protein CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats HR: Homologous recombination NHEJ: Non-homologous end joining TALEN: Transcription Activator-Like Effector Nuclease ZFN: Zinc Finger Nuclease
6. Appendices See separate file
10
https://groups.google.com/forum/#!forum/crispr
46
1: Interview transcripts 1A: Michalis Barkoulas (Imperial College; C. elegans genetics) 1B: Vlad Seitan (Kings College London; mouse genetics) 1C: Jose Gutierrez-Marcos (University of Warwick; plant genetics) 1D: Peter Sarkies (Imperial College; C. elegans genetics) 1E: Dan Wrister (TheL4b biohack lab, Los Angeles) 1F: Marcus Noyes (NYU University; ZFN expert)
2: Survey results 3: Ethics form
7. Bibliography BERRY, J. W. 1999. Emics and Etics: A Symbiotic Conception. Culture & Psychology, 5, 165-171. BODMER, W. 1985. The public understanding of science. London: The Royal Society. BOURDIEU, P. 1986. The forms of capital. In: RICHARDSON, J. E. (ed.) Handbook of Theory and Research for the Sociology of Education. New York: Greenwood. CARROLL, D. 2011. Genome engineering with zinc-finger nucleases. Genetics, 188, 773-82. DAVIES, L., FRADERA, R., RIESCH, H. & LAKEMAN-FRASER, P. 2016. Surveying the citizen science landscape: an exploration of the design, delivery and impact of citizen science through the lens of the Open Air Laboratories (OPAL) programme. BMC Ecol, 16 Suppl 1, 17. DOUDNA, J. A. & CHARPENTIER, E. 2014. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science, 346, 1258096. DVORSKY, G. 2015. Gene-Edited Dogs With Jacked-Up Muscles Are a World's First. Gizmodo.
47
EISENSTEIN, E. L. 2002. An Unacknowledged Revolution Revisited. The American Historical Review, 107, 87-105. FIELDS, S. 2001. The interplay of biology and technology. Proc Natl Acad Sci U S A, 98, 10051-4. GREEN, G. & REIN, M. 2013. Building research capital to facilitate research. Health Res Policy Syst, 11, 12. HARRIS, M. 1976. History and Significance of the EMIC/ETIC Distinction. Annual Review of Anthropology, 5, 329-350. HEGELUND, A. 2005. Objectivity and subjectivity in the ethnographic method. Qual Health Res, 15, 647-68. IGEM. 2016. iGEM Synthetic Biology [Online]. Available: http://igem.org/ [Accessed 07 August 2016]. KIM, H. & KIM, J. S. 2014. A guide to genome engineering with programmable nucleases. Nat Rev Genet, 15, 321-34. KNAPTON, S. 2015. China shocks world by genetically engineering human embryos. The Telegraph [Online]. Available: http://www.telegraph.co.uk/news/science/11558305/China-shocksworld-by-genetically-engineering-human-embryos.html [Accessed 06 August 2016]. KNORR-CETINA, K. 1999. Epistemic Cultures: How the Sciences Make Knowledge, Cambridge, MA, Harvard University Press. KUHN, T. S. 1962a. Normal Science as Puzzle Solving. The Structure of Scientific Revolutions. Chicago: University of Chicago Press. KUHN, T. S. 1962b. The Structure of Scientific Revolutions, Chicago, University of Chicago Press. KUZHABEKOVA, A. & KUZMA, J. 2014. Mapping the emerging field of genome editing. Technology Analysis & Strategic Management, 26, 321-352. LATOUR, B. & WOOLGAR, S. 1979. Laboratory Life: The Construction of Scientific Facts, Princeton, New Jersey, Princeton University Press. LEDFORD, H. 2015. CRISPR, the disruptor. Nature, 522, 20-4.
48
LEYDESDORFF, L. & WARD, J. 2005. Science shops: a kaleidoscope of science–society collaborations in Europe. Public Understanding of Science, 14, 353-372. LIANG, P., XU, Y., ZHANG, X., DING, C., HUANG, R., ZHANG, Z., LV, J., XIE, X., CHEN, Y., LI, Y., SUN, Y., BAI, Y., SONGYANG, Z., MA, W., ZHOU, C. & HUANG, J. 2015. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell, 6, 363-72. LUNAU, K. 2016. CRISPR ‘democratizing’ genetics, medicine. Genetic Literacy Project [Online]. Available from: https://www.geneticliteracyproject.org/2016/03/25/crispr-democratizinggenetics-medicine/ [Accessed March 25, 2016 2016]. MERTON, R. K. 1968. The Matthew Effect in Science: The reward and communication systems of science are considered. Science, 159, 56-63. MERTON, R. K. 1973. The normative structure of science. The Sociology of Science: Theoretical and Empirical Investigations. Chicago: University of Chicago Press. POPPER, K. 1970. Normal Science and its Dangers. In: LAKATOS, I. & MUSGRAVE, A. (eds.) Criticism and the Growth of Knowledge : Proceedings of the International Colloquium in the Philosophy of Science, London 1965. Cambridge: Cambridge University Press. ROBERTSON, M. 1980. Biology in the 1980s, plus or minus a decade. Nature, 285, 358-359. SCOTT, C. T. 2005. The zinc finger nuclease monopoly. Nat Biotechnol, 23, 915-8. SEYFRIED, G., PEI, L. & SCHMIDT, M. 2014. European do-it-yourself (DIY) biology: beyond the hope, hype and horror. Bioessays, 36, 548-51. SHALEM, O., SANJANA, N. E. & ZHANG, F. 2015. High-throughput functional genomics using CRISPRCas9. Nat Rev Genet, 16, 299-311. SHUMAN, H. A. & SILHAVY, T. J. 2003. The art and design of genetic screens: Escherichia coli. Nat Rev Genet, 4, 419-31. SINGH, P., SCHIMENTI, J. C. & BOLCUN-FILAS, E. 2015. A mouse geneticist's practical guide to CRISPR applications. Genetics, 199, 1-15. SKARNES, W. C. 2015. Is mouse embryonic stem cell technology obsolete? Genome Biol, 16, 109. 49
ST JOHNSTON, D. 2002. The art and design of genetic screens: Drosophila melanogaster. Nat Rev Genet, 3, 176-88. THELAB. 2016. THELAB: LA's Hub for Citizen Science and DIYBio [Online]. Available: http://www.thel4b.com/ [Accessed 25 July 2016]. THOMAS, K. R. & CAPECCHI, M. R. 1987. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, 51, 503-12. TIMES HIGHER EDUCATION. 2016. Available: https://www.timeshighereducation.com/worlduniversity-rankings/2016/world-ranking [Accessed 07 August 2016]. TOWNES, C. H. 1983. Science, technology, and invention: Their progress and interactions. Proc Natl Acad Sci U S A, 80, 7679-83. TRAVIS, J. 2015. Making the cut. Science, 350, 1456-7. TRAWEEK, S. 1988. Beamtimes and Lifetimes: The World of High Energy Physicists, London, Harvard University Press. WANG, H., YANG, H., SHIVALILA, C. S., DAWLATY, M. M., CHENG, A. W., ZHANG, F. & JAENISCH, R. 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Casmediated genome engineering. Cell, 153, 910-8. ZILSEL, E. 1945. The Genesis of the Concept of Scientific Progress. Journal of the History of Ideas, 6, 325-349. ZOONIVERSE. 2016. Available: https://www.zooniverse.org/ [Accessed 08 August 2016].
50