Table of Contents:
1. Introduction………………………………………………………………………………………………………………..1
2. Operation Principles…………………………………………………………………………………………………1-3
a. CRISPR/Cas9…………………………………………………………………………………………………1-2
b. Gene Knocking out in Laboratory…………………………………………………………………….2
c. Gene Drives…………………………………………………………………………………………………….3
3. Possible Applications……………………………………………………………………………………………….3-4
4. Evaluation of Gene Drives ………………………………………………………………………………………..4-5
5. Feasibility and Expected Completion Time………………………………………………………………….5
6. Gene drives in Relation to SDGs………………………………………………………………………………….5
7. Conclusion ………………………………………………………………………………………………………………….6
8. Sources……………………………………………………………………………………………………………………….7
Introduction:
Gene Drives, first idea developed in the 1960s, are genetic elements which surpass the Mendelian
Inheritance rules by copying themselves on both alleles making the gene homozygote. Thus, the
drive is inherited with a probability of 100% which means that the modified gene will spread rapidly
through the targeted population (also called a selfish gene) and establishes a change in the
Phenotype or Genotype of the whole population. The shorter the lifespan of individuals of a species,
the faster the gene drive will spread. This is called population engineering.
Gene drives are mainly proposed for use in insects (e.g. Malaria, Lyme and Zika carriers) to extinct
disease vectors, eradicate invasive species threatening a system and eradicate pests that cost
farmers around the world billions of dollars. In this research I am going to discuss the basics of the
CRISPR/Cas9 system, the ethical concerns of gene drives, its risks and advantages and the impact on
the Sustainable Development goals of the UN.
Operation Principles:
The main factor making Gene drives possible is the Crispr/Cas9 gene scissor. The Crispr/Cas9 system
(Clustered Regularly Interspaced Short Palindromic Repeats) originally was discovered as a bacterial
adaptive immune system against viruses and caused huge excitement in the gene engineering
community due to its low costs and simpler operation principles than other techniques.
a. CRISPR/Cas9:
When a bacterium is infected with a virus and survives the infection it takes a part of the DNA
inserted by the virus and inserts it in so called spacer sequences in the Crispr locus. The Crispr locus
consists of code for Cas enzymes which are either Nuclease (cut DNA backbone) or Helicases (cut
hydrogen bonds between bases) and Spacer DNAs (where viral genomes get inserted) with Crispr
repeats in between (DNA which sequences which just repeat themselves).
This step spacer Acquisition is followed by crRNA Processing. When the same virus injects its genes
into the cell again, RNA transcription of Crispr locus occurs. An mRNA of the CRISPR locus is sent to
the “intruder genome” and the mRNA is chopped into crRNAs (viral genome and RNA repeats serving
as guide RNAs) pieces which will fuse with the Cas9 protein and eventually will modify the intruder
genome. There a 3 types of crRNA processing of which the 2nd is the most important:
1. Crispr repeats form loops and mRNA is cut by either Cas6e or Cas6f
2. tracrRNA (transactivating Crispr RNA) binds to Spacer and DNA repeat -tracrRNA is used by
Cas9 protein later as a handle and mRNA is chopped into pieces of crRNA (tracrRNA + Spacer
+ repeat)
3. mRNA is chopped into spacer DNA and DNA repeats only
Then follows Interference in which each crRNA merge with a Cas9 protein which will bind to the viral
DNA. The complex of Cas9, Cas Genes and crRNAs which are used as guide RNAs (spacer RNA is
complementary to a piece in the viral genome →base pairing) will bind to complementary sequence
and PAM sequence (protospacer adjacent motif) which is about 2-6 bp downstream and is required
for Cas nucleases to cut viral DNA and increases specificity of recognition of DNA sequence (PAM
sequence is not used in Type 3 of crRNA processing). Basically, the PAM sequence is only existent in
the target DNA, so the Cas Protein can differ a Viral DNA from its own and won’t cut the DNA of the
bacterium. Then, a Cas enzyme cuts the viral DNA and the infection is prevented. In the first Type a
cas3 enzyme cuts out PAM and adjacent sequence after a Cas cascade which is not understood yet.
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In the second case the Cas9 enzyme causes a double strand brake of the DNA and in the 3rd one
another Cas cascade which is not thoroughly understood either causes the genome to be cut out.
To sum up the Crispr/Cas9 system works as an adaptive immune system for bacteria. It stores
information from the viral DNA and uses them later to protect itself.
b. Gene knocking out in laboratory:
To inactivate a gene in an organism, scientists insert a plasmid (an extrachromosomal ring of DNA,
usually several thousand nucleotides long, found in bacteria, independently replicable, easy to
genetically modify and thus used as vectors to modify genes in organisms) into the Inner cell mass of
pluripotent stem cells. The plasmid, containing the required CRISPR/Cas9 genes, code for the Cas9
protein and a DNA repair template, is either inserted per virus or by Electroporation (opening of
pores in stem cell per brief electrical pulses). When the plasmid is inserted it will undergo
transcription (polymerase II binds to promoter and activated by general transcription initiation
factors) and produce: CrRNA, tracrRNA, a Cas9 protein and the DNA repair template. Together they
form the active Cas9 complex.
The CrRNA will recognize the targeted gene and PAM
sequence in the stem cell and the Cas9 Protein causes a
double strand break. If it would not be for the DNA repair
template the DNA just would repair itself and undo the
modification. The repair template will replace targeted DNA
sequence and move into the gap caused by the double strand
break.
To sum up, gene knocking out is the insertion of a plasmid
containing the necessary information for a complex of
CrRNA, Cas9 protein and DNA repair template. Together they
will cut out DNA and replace it with the repair template
which will inactivate the gene. There also are other types of
the CRISPR/Cas9 systems but this one is by far the most
important one.
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c. Gene Drives:
CRISPR/Cas9 Systems are used
as catalysts carrying Cargo DNA
responsible for the change in
genotype (the drive). An
endonuclease (usually the Cas
protein guided by gRNA and
coded by the allele it will copy)
cuts a part of the DNA where the
drive is not located and thus
damages the DNA. Then the
natural repair system of the cell
repairs the DNA using the
template RNA carried by the
CRISPR/Cas9 system to replace
the missing DNA. Now the cell
has two copies of the same
gene.
To release the gene drive a large
number of individuals carrying
the drive has to be released into
the wild and has to pair with
non-carriers.
To sum up, every time an allele
gets inherited, (e.g. AA x Aa) the
offspring has at least 1 copy of
the dominant allele. This allele then will copy itself on the other locus and make the offspring
homozygote (AA).
Possible Applications:
Gene drives are embraced for the genetic improvements of crops, the extinction of pests as well as
the extinction of plagues like malaria. Gene drives are so called selfish genes which inactivate the
segregation law of Mendel so that they will be inherited with a 100% chance.
Possible interventions into agricultures using a drive might be the prevention of herbicide resistances
and even making some herbicides and pesticides redundant. Herbicide resistant alleles for example,
could be knocked out. By changing fertility or establishment key traits invasive species could be
thrown out of ecosystems. Agricultural goals include the protection of crops from for example
Plutella xylostella carried by the diamondback moth. The pest costs farmers all around the world $4
to $5 billion per year.
Not only agricultures are applicable aims for gene drives. Major diseases like malaria, which causes
the death of approximately 1 million people annually could be extinct by making the female
anopheles mosquito offspring infertile, thus drastically reducing the amount of anopheles mosquito
in wild nature and their ability to reproduce.
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The downsides of GDTs are the serious ethical issues and that negatives aspects of the gene drives
might not be reversable. Also, the effects of gene drives, when released, are hard to determine since
not only gene drives affect an environment but the environment also changes the gene drive. This is
caused by natural selection -and mutation processes which are difficult to predict. Ethical concerns
include the questions: How do countries come to agreements on regulations of gene drives? Can the
human invade into nature in such a drastic way? What should the legal barriers be?
Evaluation of Gene Drives:
GDTs (Gene Drive Technologies) are a controversial topic between scientists. The Arguments for the
controversy can be split up into 3 sections:
1. Dealing with uncertainty of the impacts of GDTs
2. Identifying and weighing alternatives for GDTs
3. The role of the human in nature.
Experts argue that it is uncertain at which point GDs can be applied, since we will never be able to
fully understand an ecosystem due to an unlimited number of factors influencing the system. Thus, it
is difficult to determine at which point of not-knowing one knows enough to restrict and predict
possible failures of the GD, since the intervention is not reversable. So, from when on can we be sure
that such a drastic intervention in nature is safe enough to be applied?
If you take a look at the efficiency of GDs, it is obvious they outperform conventional strategies by far
(in the field of agriculture) due to their less harmful and more accurate operation method. Pesticides
and herbicides work more radically and often have negative effects acting on the health of the end
consumer due to toxins in the crop, while GDs act only on the pests. Additionally, GDTs work
independently and are not affected by pesticides or herbicides, thus allowing scientists to use them
simultaneously. This has the advantage of making several pesticides/herbicides in use, redundant
after the GDT is established.
There are several solutions for the Malaria plague in Africa. The wide spread of insecticides in indoor
spaces or the heavy expansion of the health care system are two of them. But while the expansion of
the healthcare system would cost an immense amount of money, insecticides have the disadvantage
of being toxic to not only insects but also humans. Also, many Anopheles Mosquitos develop
resistances against the toxins. In this case GDTs could offer the comfort of erasing the plague
cheaper, faster and without affecting the health of human negatively.
The probably most polarized topic in the debate is the role of humans in nature and how this defines
the dealings with GDTs. Most of all, one has to consider the suffering and death which could be
extinguished by GDTs. Diseases responsible for the death of millions could be wiped out relatively
easy without high costs. This though, stands in contrast to the fear that one day, society might use
GDTs whenever it wants to without big consideration. The human would then take the position of
the creator who shapes nature around him in any desirable way. One could change the genotype or
phenotype of a population or even extinct it, simply because he doesn’t find any favor in it which is
highly questionable.
Nature is something which stays in balance, develops and doesn’t destroy itself. But humanity threw
that system out of balance. Every year 10 million hectares worth of forest get cleared (UN
approximates), the massive pollution of oceans causes an uncountable number of deaths of marine
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animals and birds and the global climate change is progressing etc. Everyday 150 species are ceasing
which since the Dinosauria’s made an exit this is the highest level of species extinction the world has
seen. When you compare the effects of GDTs to that, they seem insignificant. The positive aspects of
GDs might overweigh the negative ones. Millions of deaths could be saved and agriculture could be
pushed into another level of efficiency while the possibility exists that in the distant future the
technology could be used as a tool to shape nature to simply please.
Feasibility and Expected Completion Time:
Gene drives will change the manner in which science reacts to nature and eventually will open us the
door to an era in which we will be able to shape the ecosystems around us as gravitating as we
desire. The discovery of the CRISPR/Cas9 system in 2013 boosted the potential of drives enormously
due to its simple and effective applicability. But still, gene drives face serious issues like the
reversibility of the intervention in to the gene pool of a species, ignoring the ethical concerns.
Gene drives are far from an adequate application and are still in the early years of development as a
study of SPAGES (GeneTip_Endbericht.pdf, p. 19) reveals. If you want to release individuals of a
species carrying a gene drive into an ecosystem, one has to understand many factors of the
ecosystem, which is a difficult task itself, to predict the possible effects. The areas of ecology which
have to be acknowledged are the physiological part (Organelles, cells, etc.) and the ecological part
(Individuals, populations, ecosystems, landscapes, biomes and biospheres). Then you have to predict
possible external influences from e.g., humans or invading species. For a sufficient evaluation of the
dangers and possible influencing factors of gene drives, more criteria are required. There simply is
not enough data so far. This considering, gene drives are far from being used in wild nature in near
future.
Gene Drives in Relation with SDGs:
GDTs will have impact on the pathway to achieve the 2nd (Zero hunger), 3rd (Good health and wellbeing) and 15th and 16th (Life below water and on land) SDGs.
Gene drives will have a considerable impact on the 2nd goal since agriculture is one of their major
targets, allowing more efficient and cheaper food production due to pesticide/herbicide replacement
and less pest threats. The 3rd goal which is “Good health and well-being” obviously is positively
affected by aiming the eradication of major disease vectors such as the anopheles mosquito. GDTs
also will have an effect which is difficult to determine on all life on land and under water. The role of
the human will further shift into a position in which he has more control over nature and biodiversity
than ever before in history. The question which stays open, is how heavy and negatively or positively
GDTs will influence the 14th and 15th goal. While biodiversity will be reduced, invasive species could
be “thrown” out of an ecosystem and help it recover. The role of GDTs in this case stays unclear.
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Conclusion:
To sum up, GDTs could be used to extinguish or change the genotype of disease vectors, saving
millions of lives, pests, harmful for crops, to make agriculture more efficient and cheaper, and
invasive species, threatening an ecosystem. To evaluate, whether or not use GDs, I listed up all pro
and contra arguments. As mentioned, it is difficult to determine, when we know enough factors of an
ecosystem to predict the impacts of the GD. When we change an ecosystem in such a drastic way, we
shouldn’t be only 90% or 95% sure that the GD will work, because the GD can’t be reversed.
GDs clearly outperform conventional strategies in agriculture as well as in the controlling of disease
vectors by being health unaffecting to humans and the complimentary use next to pesticides (GDs
don’t have health impact on the humans like e.g. pesticides or herbicides and can be used in a
addition to pesticides or herbicides to increase efficiency). With GDs the fight with not only malaria
but also many other insect transmitted diseases like Zika, Dengue, Yellow Fever and Chikungunya
could come to an end.
I personally favor the use of Gene Drives because of the many lives they could save. The interest of
an animal transmitting malaria in this case simply weighs less than the interest of a human dying
from malaria. Thus, one has to accept the negative aspects which come with the use of gene drives
including a reduction of biodiversity and the uncertainty which is related to the release of GDTs.
Gene Drives inevitably will change how we think of nature and how we react to it and push us more
and more into a position in which we can shape our surroundings in any imaginable way, which, to
note we already do by for example causing the climate change.
I think Gene Drives shouldn’t be only decided by Governments. Local communities influenced by the
interventions should also be engaged into the debate since they are being affected the most. The
public should be informed about gene drives on a base on which one can form a reasonable opinion.
Maybe the public even should be engaged into the finding of the process of making a decision
whether the gene drives should be applicated or not. But because ethical concerns are far more
important than to be evaluated by only a local community, national or even international
agreements should be made. This method is called public deliberation. The process of such a thing is
unclear so far and would reach far beyond this project.
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