Genetic Engineering: Reading, Writing and Editing Genes [1 ed.] 9783658324025, 9783658324032

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Röbbe Wünschiers

Genetic Engineering Reading, Writing and Editing Genes

essentials

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Springer essentials provide up-to-date knowledge in a concentrated form. They aim to deliver the essence of what counts as "state-of-the-art" in the current academic discussion or in practice. With their quick, uncomplicated and comprehensible information, essentials provide: • an introduction to a current issue within your field of expertis • an introduction to a new topic of interest • an insight, in order to be able to join in the discussion on a particular topic Available in electronic and printed format, the books present expert knowledge from Springer specialist authors in a compact form. They are particularly suitable for use as eBooks on tablet PCs, eBook readers and smartphones. Springer essentials form modules of knowledge from the areas economics, social sciences and humanities, technology and natural sciences, as well as from medicine, psychology and health professions, written by renowned Springer-authors across many disciplines.

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Röbbe Wünschiers

Genetic Engineering Reading, Writing and Editing Genes

Röbbe Wünschiers University of Applied Sciences Mittweida Mittweida, Germany

ISSN 2197-6708 ISSN 2197-6716 (electronic) essentials ISSN 2731-3107 ISSN 2731-3115 (electronic) Springer essentials ISBN 978-3-658-32402-5 ISBN 978-3-658-32403-2 (eBook) https://doi.org/10.1007/978-3-658-32403-2 © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 The translation was done with the help of artificial intelligence (machine translation by the service DeepL.com). A subsequent human revision was done primarily in terms of content. This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Responsible Editor: Sarah Koch This Springer imprint is published by the registered company Springer Fachmedien Wiesbaden GmbH part of Springer Nature. The registered company address is: Abraham-Lincoln-Str. 46, 65189 Wiesbaden, Germany

What You Can Find in This essential

• An insight into what reading and editing the human genome means to us. • An overview about marketing genetic information and how the environment and life style are affecting it. • An outlook on how the writing of genetic information is expected to revolutionize biotechnology and how everyone can contribute to it.

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Preface

Dear Reader Just as genetic engineering has revolutionized our everyday life and science is in danger of drowning in data, the Internet is also revolutionizing our everyday life. Actually, one could be informed about everything, alone, there is not enough time and sometimes not the right access. With this essential I would like to offer a small insight into the fascinating and controversial world of genetic engineering. Kant asks: What can I know?—What should I do?—What can I hope?—What is man?— I would like to contribute to the first question. The second question you must decide for yourself—and probably on a case-by-case basis, because we must learn to think and argue in colors, not black and white. I hope that your thoughts on genetic engineering will not be guided by ideologies and feuds, but by a witty weighing up of the benefits and risks. I also hope that you will keep putting your decisions to the test. Yes, Homo sapiens is a wise man, but he is also a tinkerer, one with a larger ecological footprint than a sauropod, which has also released a lot of greenhouse gas.1 To the Content This essential should serve as an introduction to a contemporary public discussion on genetic engineering and its application.2 It may be less essential than an essence. It contains roughly 12,200 words. An average reader reads 300 words per minute. 1 Wilkinson

DM, Nisbet EG, Ruxton GD (2012) Could methane produced by sauropod dinosaurs have helped drive Mesozoic climate warmth? Curr Biol 22:R292–R293. doi: 10.1016/ j.cub.2012.03.042. 2 The author is grateful that parts of his inquiries were made possible by funds from the “Innovative Hochschule” program, sub-project Saxony5 , funded by the Federal Ministry of Research and Education. vii

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The rest is brain jogging. This book is accompanied by a website on gene-genomegesellschaft.de. What it is all About Genetic engineering has reached the public—not only in the form of products from the pharmaceutical, food and detergent industries, for example, but also as a political issue. At the latest since the announcement in 2018 that CRISPR/Cas gene edited twins were born in China or the announcement in 2017 that the Bayer Group acquires the seed and crop protection company Monsanto, almost everyone is aware of at least two applications of genetic engineering: gene therapy, in this case even in the germline of human embryos, and the design of glyphosate-resistant Roundup Ready® plants. The debate on genetic engineering has spread from the science section of newspapers to the political and business sections and even to the arts pages. And this is very good. Nevertheless, the public discussion on genetic engineering seems to me to be suffering from a number of problems. For example, it is often based on old pictures and methods and is usually conducted in black and white. A strict distinction must also be made between the technology itself, its application and its marketing. All too often, the extent to which genetic engineering already penetrates and will penetrate our everyday life is also neglected. Although genetic engineering is already deeply rooted in our everyday consumer lives, the near future will offer even more personal contacts with genetic engineering: Should I undergo gene therapy; should I clone my deceased dog; should I select my offspring by carefully selecting my eggs or sperm? Röbbe Wünschiers

Contents

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2 Reading the Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3 Editing the Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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4 Writing the Genome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5 Marketing Genetic Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6 Genetics and the Environment: Epigenetics . . . . . . . . . . . . . . . . . . . . . . .

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7 Citizen Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Introduction

DNA (deoxyribonucleic acid) is a macromolecule that is built from a sequence of building blocks, the nucleotides. In the case of humans, there are about 3.2 billion nucleotides. In cells, the genetic information is divided among several chromosomes. Every human body cell contains two times 23 chromosomes, each from the father and the mother. If the DNA of the 46 chromosomes of a single human cell were joined together to form a thread, it would be about two meters long. The DNA of all the cells of a human being would reach from the earth to the sun and back about four times. At around 9 billion km, this corresponds roughly to the orbit of the planet Saturn around the sun. The genetic information on the chromosomes is distributed among genes. The totality of all genes of a living being is generally referred to as the genome or individually as the genotype. A gene can be understood as a genetic information package that codes for a trait, for example, the blood group. The totality of all traits of a living being makes up its phenotype, its appearance. For example, since there are several blood groups (O, A, B, AB), there must be several variants of the responsible gene, which we call alleles. From each gene, we carry one maternal and one paternal allele. Some traits, such as eye color, involve many genes. The basis of any diagnostic analysis and genetic engineering work is a deep understanding of the function of a particular section of the genome. In the early days, this was only possible in a very rough way. So-called genetic markers were associated with phenotypic manifestations such as diseases or other characteristics. These markers were initially not nucleotide sequences (DNA sequences), but rather physical observations, such as the fact that DNA breaks down into fragments of varying size after treatment with a DNA-cutting enzyme (restriction enzyme). The size and distribution of the fragments could be measured and correlated with traits. Today, we can read the entire genetic material (the genome) of a living being, from bacteria to humans (see Chap. 2). © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. Wünschiers, Genetic Engineering, Springer essentials, https://doi.org/10.1007/978-3-658-32403-2_1

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Introduction

Closely related to DNA sequencing is also the desire to specifically modify the DNA sequence. Corresponding methods have existed since the beginnings of genetic engineering in the 1970s. They have become more and more precise over time. The current climax is the CRISPR/Cas system, which was introduced in the 2000s and is widely used as a molecular scissor in gene editing, with the help of which the genetic material can be specifically altered (see Chap. 3). But even today, genetic engineering has become an integral part of our everyday life. There are very obvious applications, for example, when food is labeled to contain genetically modified soya oil. Experts in the food industry estimate that in around 70% of the food on the market, genetic engineering is used without the need for labeling. In addition to accidental and technically unavoidable traces of approved genetically modified organisms (GMO), these are mainly additives such as vitamins, amino acids, enzymes and other auxiliary substances produced with the help of genetically modified microorganisms. Hardly any fruit juice can do without optimized and genetically produced enzymes. This applies equally to medicines or additives in detergents or toothpaste. The methodological repertoire of classical plant breeding includes somatic hybridization. This form of “cell crossing” is also possible across genus boundaries. The result can, for example, be a Pomato (a cross between a tomato and a potato) or a virus-resistant asparagus variety. This form of fusion of two genomes does not fall under the regulation of genetic engineering. But the latest methods go even further. In 2010, a team of scientists led by the US biochemist Craig Venter presented the first organism whose genome was largely produced chemically in the laboratory and “booted” in another cell. We are now able to write genetic information on a large scale (see Chap. 4). The methods developed in the course of this process mark the beginning of a new era in genetic engineering, known in scientific circles as synthetic biology, but which society has not yet taken much notice of.

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Reading the Genome

DNA sequencing using the method developed by Frederick Sanger in the 1970s is an important building block that has driven genetic engineering forward. Sanger sequencing (also called chain termination method) is the first generation sequencing method. While in the initial phase, it was possible to analyze around 10,000 nucleotides per day and device, modern capillary sequencers of the 2000s were able to analyze around one million nucleotides. Sanger sequencing was predominant on the market for 30 years because, until recently, it allowed for the largest reading length of approximately 800 nucleotides. Approximately 800 nucleotides? However, the human genome contains 3.2 billion nucleotides. How can this be decoded? Imagine the genome as a book. With Sanger sequencing, the first 800 characters can be read in about three hours. In another three hours, the next 800 characters can be read, and so on. To be faster, we cut the book into many snippets of 800 characters each and read them in parallel in three hours. But how do we know at the end which character string follows which character string? We do not! That is why we need at least two books, each of which is snipped in different places. For example, one book every 800 characters starting from character one and another book every 800 characters starting from character 200. Then we get overlapping snippets and can reconstruct the story of the original book. This is how genome sequencing works. The 800 nucleotides long sequence fragments (reads) must be assembled to form the entire genome. This assembly is an important step and works the better the longer the reads are and the more genome copies are sequenced. So, when we talk about a genome having been sequenced, in reality more than twenty copies have usually been analyzed. In 1996, the Swedish biochemist Pål Nyrén from the Royal Institute of Technology in Stockholm/Sweden developed the so-called pyrosequencing together with his PhD student Mostafa Ronaghi. It has nothing to do with fireworks,

© Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. Wünschiers, Genetic Engineering, Springer essentials, https://doi.org/10.1007/978-3-658-32403-2_2

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Reading the Genome

but rather with the detection of the molecule pyrophosphate (also called diphosphate). Pyrophosphate is released when the DNA polymerase is active, as in Sanger sequencing, which is the key enzyme of sequence analysis. A light signal is then released via several enzymatic steps. The revolutionary thing about this second-generation technology is the massive parallelization and acceleration of the reading process. Pyrosequencing thus provides the result almost in real time. However, the length of the DNA sequences (reads) is limited to around 100 nucleotides. It depends on the scientific question whether this length is sufficient. History was written by the Swedish company 454 in 2008, when it analyzed and presented the complete genome of the co-discoverer of the DNA structure and Nobel Prize laureate James Watson1 with its Genome Sequencer FLX pyrosequencer.2 Although the project can certainly be seen as a promotion for their technology, 454 showed that within four months—with a handful of scientists and a little less than 1.5 million US$—a complete human genome can be deciphered. When the results of Project Jim were presented to the public, Watson made only one condition: He did not want information about his variant of the apoE gene (which encodes apolipoprotein E that plays an important role in fat metabolism) to be made public. This is because one variant of this gene is associated with the early onset of Alzheimer’s—Watson was 79 years old at the time of his sequencing. It can be said that the Project Jim was the first major sequencing project of a new generation of high-throughput real-time sequencers—and the second fully sequenced human genome. This is because in 2007, the American biochemist Craig Venter presented his genome sequence.3 It was sequenced using the Sanger method on high-throughput machines called ABI 3730xl from Applied Biosystems. This project took about seven years and cost about 100 million US$. Based on his genetic data, Venter also published his autobiography in 2008—the first autobiography to relate life events to gene variants.4 Both projects took advantage of the fact that a first draft of an incomplete sequence of the human genome had been published in 2000. This data helped both teams to merge the reads. Today, over 400,000 individual human genomes, even those of Neanderthal and Denisovan man, have been deciphered and the cost per genome is less than US$ 1000. 1 Watson

JD (1968) The Double Helix. Weidenfeld and Nicolson, London. DA, Srinivasan M, Egholm M et al. (2008) The complete genome of an individual by massively parallel DNA sequencing. Nature 452:872–876. https://doi.org/10.1038/nature 06884. 3 Levy S, Sutton G, Ng PC et al. (2007) The diploid genome sequence of an individual human. PLoS Biology 5:e254. https://doi.org/10.1371/journal.pbio.0050254. 4 Venter JC (2008) A Life Decoded: My Genome: My Life. Penguin Books, New York. 2 Wheeler

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Reading the Genome

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In the 2000s, numerous new sequencing methods were developed and brought to market maturity. Sequencing machines became more compact, cheaper and able to analyze more nucleotides per time and device. Progress in microfluidics was a major contributor to this development. Modern sequencing machines are about the size of a laser printer, cost around US$ 40,000 and can analyze 100 billion nucleotides per day. In 2013, the UK company Oxford Nanopore Technologies heralded a new era in genome analysis when, during a conference in Marco Island, Florida/USA, it presented a sequencing machine that fits in the palm of your hand and can be connected to the USB port of a standard computer: the MinION. This thirdgeneration sequencing machine, which is being marketed since 2014, is based on nanopores in a membrane through which the DNA molecule is pulled. This is associated with a change in the electric current flowing through the pore, which is in turn measured. What is revolutionary is, on the one hand, the compactness of the device and, on the other hand—and this is what is special—that sequences of several tens of thousands of nucleotides can be read. Mobile sequencing with nanopores will not only revolutionize genetic diagnostics. The method can already be used to analyze RNA and in future it will certainly also be possible to analyze proteins. Nanopore technology could then be used to identify individual proteins or protein modifications (posttranslational modifications) and analyze DNA–protein interactions. Since the MinION is a hand-held device, biological samples no longer have to be sent to a service provider, but can be analyzed in real time at the patients’ bedside, in the field, you name it. It is very conceivable that in the near future small sequencing machines in educational science kits for children will be sold in toy shops. This is a prospect that I will deal with in Chap. 7. The insights we can gain from reading the genetic information of humans who died a long time ago are fascinating. Pioneering work in this field of paleogenetics (genetics based on ancient DNA) is being carried out by the Swedish evolutionary geneticist and director of Department of Evolutionary Genetics at the Max Planck Institute for Evolutionary Anthropology in Leipzig/Germany, Svante Pääbo.5 He developed the methodological tools to isolate and sequence DNA from ancient biological material such as mummies or bone finds. Paleogenetics attracted a lot of attention with the publication of a large part of the DNA sequence of a Neanderthal man at least 35,000 years old and a Denisova man at least 30,000 years old in 2010. The results of the sequence analysis brought new light

5 Pääbo

S (2015) Neanderthal Man: In Search of Lost Genomes. Basic Books, New York.

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into the evolutionary and migration history of modern man. Around 4–7% Neanderthal or Denisovan DNA in our genome tells its own story of more or less romantic evenings between Homo sapiens and Homo neanderthalensis around the campfire. Only recently, the genome of a girl who died more than 50,000 years ago was analyzed, from which it was revealed that her mother was a Neanderthal and her father a Denisovan.6 The genome of the “Iceman”, better known as Ötzi,7 discovered in the Ötztal Alps in 1991, was also decoded and presented to the public in 2012. The genetic material from the Copper Age, which is at least 5300 years old, tells us, for example, something about the geographical origin of his ancestors (Corsica), his eye color (brown), his blood group (O), an experienced Borrelia infection, his lactose intolerance and a predisposition for the disease of his coronary vessels. How is all this possible? A decisive contribution in terms of diagnostics is made by so-called association studies, which examine which genetic information is associated with which disease pattern. These associations can be very simple: a mutation in a gene is responsible for a disease. Currently, more than 2700 such monogenic diseases are known. However, the situation is usually more difficult because there are several genetic variants (alleles) of such genes. Cystic fibrosis (a disease characterized by viscous bodily secretions) is a monogenic disease, which affects every 3000th newborn child in Northern Europe. However, more than 1000 variants of the CFTR gene (cystic fibrosis transmembrane conductance regulator) are known. It is approximately 250,000 nucleotides long and codes for a protein of 1480 amino acids in length. There is plenty of room for mutations. However, most diseases are polygenic and are therefore caused or influenced in their expression by several genes. The type of mutation in the genes or alleles can also be diverse and can range from duplications, insertions or deletions of nucleotides to single nucleotide variations (SNPs). Of the 3.2 billion nucleotides in the human genome, over 320,000 positions that have been clinically tested are known to be associated with diseases. It would therefore be sufficient to analyze these 320,000 positions in the genome in order to be able to make statements about the state of health and risk of disease. In the age of self-measurement and self-optimization, this generates a market. The American company 23andMe, founded in 2006, offers genetic analyses to anyone for US$ 199. After sending in a saliva sample, the company uses it to 6 Slon

V, Mafessoni F, Vernot B et al. (2018) The genome of the offspring of a Neanderthal mother and a Denisovan father. Nature 561:113–116. https://doi.org/10.1038/s41586-0180455-x. 7 Fleckinger A (2018) Ötzi, the Iceman. Folio Verlag, Bozen.

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isolate and analyze DNA from oral saliva cells. Even if it is not explicitly stated, it can be assumed that only certain areas of the genetic material are analyzed. Based on this data, the customer receives (a) an ancestry report including his relationship to Neanderthal man; (b) a trait report on health risks such as breast cancer and Parkinson’s disease; (c) a wellness report, including a genetically predicted optimal weight (the so-called genetic weight) or the presence of lactose intolerance; (d) a status report on thirty different phenotypic traits such as the consistency of earwax, eye color or tendency to early or late onset of disease; and (e) a hereditary carrier status report in respect of 43 diseases such as sickle cell anemia. The explosive aspect of private genome analysis lays in the fact that the customer is left alone with the data he or she retrieves from his or her personal website. This violates, for example, the German Genetic Diagnostics Act (Deutsches Gendiagnostikgesetz), according to which only a physician with proven expertise is allowed to disclose and explain the findings to the patient. And there is a reason for this: for example, genetic detection of Huntington’s disease (a hereditary disease of the brain) has a high predictive power, whereas a detected mutation in the BRCA1 gene associated with breast cancer has only a low predictive power with regard to the onset of the disease. This is due to the fact that often not only one but several genetic components or even epigenetic and environmental factors (see Chap. 6) play a role. The German Genetic Diagnostics Act came into force in 2010 and regulates “genetic testing for medical purposes, for clarifying parentage, as well as in the insurance sector and in working life.” The law regulates the requirements for genetic analyses and the use of the data obtained from them. This is intended to “prevent discrimination on the basis of genetic traits” and “to safeguard the state’s obligation to respect and protect human dignity and the right to informational self-determination.” The law distinguishes between diagnostic and predictive (prognostic) genetic tests. A diagnostic examination serves to clarify an existing disease or health disorder and to determine genetic traits, which, together with other influences (such as interaction with drugs), can trigger the onset of a disease or disorder. Such an examination may only be carried out by medical doctors and the results may only be disclosed to the patient by medical doctors. A predictive examination serves to clarify a possible future disease or health disorder and to clarify the presence of a genetic disposition that could be passed on to descendants. Predictive genetic testing may only be carried out by medical specialists in human genetics or medical doctors who are specially qualified in human genetic testing. In addition, the patient must agree to the genetic examination and be fully informed beforehand, including about the significance for

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his or her future life. The major difference between the currently prevailing diagnostics, which focuses on certain measurements, and genome analyses (and also transcriptome analyses; see below) is the density of information generated about a patient. Thus, a genome-wide analysis does not represent a punctual, but rather an ongoing encroachment on information rights, since more and more information can gradually be obtained from the stored genetic data. This applies regardless of whether the sequencing had a diagnostic (acute) or predictive reason. Here, it is important to clarify that the patient may learn things about his or her genetic makeup that may make him or her feel more uncertain than being helpful. Whereas in the past individual genes, sections of genes or entire genomes, i.e., DNA, were analyzed in diagnostics, the latest trend is toward RNA. RNA diagnostics (or transcriptome diagnostics, also known as RNA-Seq) is used to determine which genes are active. It is therefore the “living” part of the genome. This form of diagnostics cannot only be performed for individual genes but also for the entire genome. While the information of a single gene expressed by the cell is called a transcript, the entirety of all transcripts in a cell is called a transcriptome. Two studies published in 2017 showed that the method of transcriptome sequencing was able to diagnose diseases that were not identified by standard genetic diagnostics. In addition, new candidate genes for the respective diseases were found. With the help of such additional information, a diagnostic test can be gradually improved by increasing the number of examinations. In both senses, diagnostic and predictive, prenatal genetic analyses can also be performed. This form of diagnostics has developed rapidly in recent years, not least due to the increase in artificial insemination. Louise Joy Brown was born on July 25, 1978 and was the first person to be conceived using in vitro fertilization (IVF). The first German child conceived using IVF was born on April 16, 1982. The number of IVFs in Germany has multiplied from 742 treatments in 1982 to around 90,000 completed treatments in 2016. In more than half of the cases, the male sperm is injected into the egg cell with a kind of syringe; in about a quarter of the cases, the sperm had been frozen beforehand. The latter is increasingly being offered to female employees of companies such as Apple or Facebook: freezing their eggs in order to postpone motherhood to times after a successful career and establishment in the company (or better another?). This procedure is called social freezing and is, unlike egg donation, not prohibited in Germany. For women who wish to have children after the menopause, it is a legal way to conceive a child. In 2015, a 65-year-old retired Berlin/Germany woman gave birth to quadruplets after artificial insemination. The number four alone shows that the implantation of the fertilized eggs did not take place in Germany, where the number is limited to a maximum of three embryos, but in this case in Ukraine.

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And this would not be possible in Germany either: both sperm and egg cell came from donors. However, if the fertilization process has to be transferred from the body to a laboratory for medical reasons, the barrier for accepting further medical services is often lower. This is because if you have to undergo the physically and often emotionally stressful procedure of egg and sperm retrieval, then you want to maximize the reproductive success. The decision to have an abortion could also be made at an early stage with a genetic test—in extreme cases even before fertilization. In the field of prenatal diagnostics of embryos and fetuses, a distinction must be made between analysis during pregnancy (prenatal diagnostics) and examination within the framework of reproductive medicine, i.e., before a possible transfer of an embryo into the uterus (preimplantation diagnostics). In Germany, prenatal diagnostics, like the genetic testing of born human beings, is governed by the Genetic Diagnostics Act and therefore stipulates that a genetic analysis “may only be carried out for medical purposes and only if the examination is aimed at certain genetic traits of the embryo or fetus, which, according to the generally recognized state of science and technology, affect its health during pregnancy or after birth, or if treatment of the embryo or fetus with a medicinal product is envisaged whose effect is influenced by certain genetic traits”. The pregnant woman, in turn, must be informed and consent to the examination. On the other hand, an examination in relation to a disease that, according to the acknowledged state of scientific knowledge, may break out after the age of eighteen, is not permitted. In contrast, preimplantation diagnostics is not regulated by the German Genetic Diagnostics Act. In 2011, however, a regulation was made in the Embryo Protection Act (Embryonenschutzgesetz). In this law, preimplantation diagnostics is punishable offence and is only permitted in exceptional cases. This applies to cases in which, due to the genetic predisposition of one or both parents, there is a high risk of a serious hereditary disease for descendants. According to the legislator, a serious hereditary disease is deemed to exist if the disease “differs substantially from other hereditary diseases due to a low life expectancy or the severity of the clinical picture and poor treatability”. Genetic testing of the germ cells prior to fertilization is currently only possible to a limited extent. In so-called polar body diagnostics, the genetic material of the polar body cell is examined, which is very similar to that of the egg cell—but not identical. Since sperm or egg cells are destroyed in a state-of-the-art genetic analysis, a precise prefertilization diagnosis is currently not possible. However, this will probably change soon. Various research groups are recording initial successes in the production of germ cells from precursor stem cells. If these could be

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reproduced (cloned) in the laboratory, a more precise diagnosis would be possible. What would be conceivable effects? A genetic test prior to fertilization might result in a selection of sperm and egg cells. What are or would be the criteria? How do we separate a serious disease, which would allow selection, from a disease that causes discomfort at best? How do we deal with the probabilities that a genetic information will actually become effective during the life of the offspring? In the USA, sperm cells can already be selected by catalog: for example, from Nobel Prize laureates, blue-eyed people or successful athletes. What about intelligence or, more precisely, selection according to cognitive traits? In 2017, a genetic study of 78,308 individuals identified 336 nucleotide positions (SNPs) distributed among 22 genes that are associated with cognitive capabilities.8 In addition, these SNPs are related to Alzheimer’s disease, depression, autism and life expectancy, for example. As a rule, we would like to send our children to the best local school—why not give the offspring the best possible genome? This may all sound very far away, but the technical possibilities are already available today. Ultimately, however, there are also opportunities after birth and in advanced age to intervene in the genome therapeutically or by optimizing it in the course of gene therapy.

8 Sniekers

S, Stringer S, Watanabe K et al. (2017) Genome-wide association meta-analysis of 78,308 individuals identifies new loci and genes influencing human intelligence. Nature Genetics 49:1107–1112. https://doi.org/10.1038/ng.3869.

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Editing the Genome

There are a multitude of different methods to intervene in the genetic makeup of living beings. In general, undirected methods, which randomly cause genetic changes either via ionizing radiation or via chemicals, must be distinguished from specific or site-directed methods. The latter are gaining increasing interest and are now widely used by scientists as gene or genome editing tools. With DNA sequencing and the subsequent elucidation of the relationship between the appearance (phenotype) of an organism and its genetic material (genotype), we are no longer dependent on chance. On the contrary, one would like to intervene specifically in the genome. In August 2012, US biochemist Jennifer Doudna1 from the University of California/USA at Berkeley and French microbiologist Emmanuelle Charpentier, now director of the Max Planck Unit for the Science of Pathogens in Berlin/Germany, published a groundbreaking paper: a method for the targeted modification of a chosen DNA sequence in the genome.2 –In 2020, both received the Nobel Prize for their work. –A few months later, in February 2013, the US biochemist Feng Zhang showed how the system can be applied to human and mouse cells.3 The method is based on the CRISPR/Cas system and can cause nucleotide-precise changes in the genome as precisely as a scalpel or scissor on an surgical table. Hence, the name genetic surgery or, based on the genetic code, genome editing. Today it is clear: the system works in every living organism. The 1 Doudna

JA, Sternberg SH (2017) A Crack in Creation: Gene editing and the unthinkable power to control evolution. Houghton Mifflin Harcourt, New York. 2 Jinek M, Chylinski K, Fonfara I et al. (2012) A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337:816–821. https://doi.org/10.1126/ science.1225829. 3 Cong L, Ran FA, Cox D et al. (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823. https://doi.org/10.1126/science.1231143. © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. Wünschiers, Genetic Engineering, Springer essentials, https://doi.org/10.1007/978-3-658-32403-2_3

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procedure is so simple that in the USA, it is even sold to everyone as an experimental science kit for US$ 159 that may be used in schools (see Chap. 7). And last but not least: it is not genetic engineering—at least not in many countries. However, in Europe it is, since the ruling of the European Court of Justice on July 25, 2018 (see below). But first things first. CRISPR stands for clustered regularly interspaced short palindromic repeats. Whew, what is that? They are clustered short palindromic sequences that are repeated and interspaced at regular intervals. Simple palindromes are Otto or “sex at noon taxes”: they can be read from front and back. In genetics, a palindromic sequence indicates that it has the same sequence on the opposite strand of DNA in the opposite direction. For example, the sequence AACGTT on the opposite strand reads the same. So what does it mean that such DNA sequences occur in a genome? They were discovered in bacteria as early as 1987, when the Japanese molecular biologist Yoshizumi Ishino from Osaka University wrote in a publication4 : “an unusual [DNA] structure was found”. It was an incidental remark. The Spanish microbiologist Francisco Mojica described this unusual DNA sequence in more detail in 1993 and found out ten years later that the repeated palindromic sequences were only the spacers for DNA sequences that bear a strong resemblance to the genome of known bacterial viruses (phages). In 2002, the Dutch microbiologist Ruud Jansen proposed the name CRISPR together with Mojica. While repetitive sequences were of no interest to most scientists, research now picked up speed. In 2006, the US bioinformatician Eugene Koonin published a comprehensive study5 on CRISPR and showed for the first time how widespread it is in bacteria. He developed the hypothesis that it could be a bacterial immune system against phages. Just as our immune system “remembers” which particles (antigens) it had already come into contact with, the CRISPR region stores sequences of phages with which the bacterium or a predecessor had already had contact. Subsequently, several scientific teams reported that CRISPR-associated genes (abbreviated Cas) code for proteins that can cut DNA sequences. It is primarily thanks to the research cooperation between the scientists around Doudna and Charpentier that the complete mechanism has 4 Ishino Y, Shinagawa H, Makino K et al. (1987) Nucleotide sequence of the iap gene, respon-

sible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product. Journal Bacteriology 169:5429–5433. https://doi.org/10.1128/jb.169.12.54295433.1987. 5 Makarova KS, Grishin NV, Shabalina SA et al. (2006) A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biology Direct 1:7. https://doi.org/10.1186/1745-6150-1-7.

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been elucidated and optimized to the extent that it can be adapted to all living organisms. According to this, an RNA molecule is formed from the sequences of the spacers, which serves as a template for the Cas enzyme to cut at exactly this sequence in the genome. This RNA molecule can be generated synthetically and adapted to any sequence. This has turned a bacterial immune system into the most effective genetic engineering tool currently available; more effective, cheaper and easier to adapt than previously used systems. It is worth noting that, following the revolution in the life sciences caused by restrictive enzymes in the 1970s, a bacterial defense mechanism against phage is again causing a sensation in the 2010s. However, Herbert Boyer and Stanley Cohen have not (yet?) received a Nobel Prize for their work. Are there any problems? Yes, it is possible that the CRISPR/Cas system makes mistakes and induces changes in other parts of the genome. Minimizing this is currently an important goal of worldwide research. The number of scientific publications on CRISPR can also be seen in the number of publications on the topic: from 2005 to 2010, there were a few dozen; in 2015, there were around 1500, and in 2018, it is expected to be over 5000. Is this technology genetic engineering? This question can be answered with a resounding “yes” when you look at the process. The tool for genetic mutagenesis must be introduced into the target cell, i.e., a Cas enzyme (usually Cas9) and the RNA construct that directs the enzyme to the correct DNA position. For this purpose, the genetic sequence information is usually introduced into a DNA vector, which is then channeled into the target cell. This process is called transformation in plants and bacteria, and transfection in vertebrates such as humans, and is a genetic engineering process. However, the vector always remains separated and is not incorporated into the genome. Once expressed, the CRISPR/Cas system does its work. The DNA vector is then lost in the daughter cells of the subsequent cell division. It is not multiplied and passed on like the genetic material, but is only present transiently. It can therefore be said that the process is genetic engineering, but the product is not. However, the European Court of Justice concentrated on the process in its judgment. There is probably also certain helplessness in this judgment, as the black and white consideration of GMO versus no GMO in the CRISPR/Cas system does not go far enough. In order to do justice to the new technologies, different levels of authorization, combined with different authorization procedures, would have to be established. The approval system should be as precise as genetic engineering. A targeted point mutation, in which a nucleotide in the genome has been exchanged, cannot always be equated with the transfer of a gene from one species to another. A multistage approach would also enable smaller breeding companies or associations to place GMOs on

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the free market, simply because simpler approval procedures would reduce the financial outlay (see Chap. 5). Independently of this important regulatory issue, gene editing is used intensively, for example, in medical and breeding research. There is a gold-rush atmosphere. Black and white is also the public perception of the use of genetic engineering in agriculture. For many people, for example, ecological and conventional agriculture are mutually exclusive. Farming according to organic guidelines and using genetically modified seeds or related products seems incompatible. In their book Tomorrow’s Table,6 the US professor of plant pathology Pamela Ronald and her husband Raoul Adamchak, head of an organic student farm in Davis, California/USA, show that the two economic approaches—or philosophies?—can certainly be combined. A vivid example comes from rice cultivation. Young rice seedlings can survive for several days completely submersed. Organic rice cultivation takes advantage of this, as watering chokes off unwanted accompanying plants. However, it is important to find the right time to let the water run off again, otherwise the rice seedlings will also suffocate. Rice varieties that can survive long periods of submersion would be advantageous. Pamela Ronald has isolated a gene (the so-called SUB1 gene) from an extremely “submersion tolerant” rice variety. Built into a less tolerant rice variety, it was able to survive for eighteen days in submersion. The Sub1 gene was also successfully tested in wheat, maize and soya. Now the question arises as to why the tolerant rice variety is not cultivated immediately instead of isolating the responsible gene and genetically engineering it into other rice varieties. The keyword here is: varieties. Of all plants, there are varieties that are optimally adapted to certain regional microclimates. In plant breeding, therefore, desired traits must always be crossed into locally adapted varieties. This is not always successful and genetic engineering offers a comparatively quick solution here. The two Americans are therefore not the only ones who see an opportunity in the interaction of genetic engineering and organic farming. The renowned Research Institute of Organic Agriculture (Forschungsinstitut für biologischen Landbau, FiBL), which was founded in Switzerland in 1973, is active in research, development and consulting for organic agriculture. It currently has around 175 employees throughout Europe, who also sees opportunities, for example, in the application of gene editing in organic agriculture. Its former director, Professor Urs Niggli, is one of the world’s leading experts in the field of organic farming. He has been advocating a differentiated discussion on genetic engineering for quite some time, for which he has received a lot of criticism from organic 6 Ronald

PC, Adamchak RW (2018) Tomorrow’s Table. Oxford University Press, New York.

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farming associations. In an interview with Der Tagesspiegel,7 Niggli said: “After thirty years of debate, it would be desirable to become more objective.” One has “important problems to solve, such as the fact that we produce more than enough food, but only with huge amounts of pesticides and fertilizers.” It will be important to question entrenched ideologies and patterns of argumentation, to bring the latest findings into the discussion, to think in terms of colors rather than black and white, and to approach each other in consideration of opportunities and risks. I would like to look at one more special application of gene editing, the gene drive. Here, the CRISPR/Cas system is built into the genome of an organism, i.e., a GMO is produced. The system is then able to act in its own genome and—after fertilization—on the “new” genome. This ability is passed on to the following generations. Work is being done, for example, on mosquitoes that are immune to malaria and therefore cannot infect humans. Using the gene drive system, they pass on this immunity to wild nonimmune mosquitoes with which they cross. The system thus causes the gene drive mosquitoes to genetically modify other mosquitoes. The immunity spreads throughout the population. This is known as a mutagenic chain reaction or super-Mendelian inheritance. It must be clearly stated that a system is being created that can no longer be stopped.8 In most cases, transgenic organisms are deliberately attempted to prevent their spread in nature. The gene drive has exactly the opposite aim. Similarly, the US Pentagon Research Institute is currently researching the possibility of genetically modifying crops in the field.9 To this end, insects are to infect the plants with genetically modified viruses. These viral vectors should in turn contain a CRISPR/Cas system that modifies the plant genomes. Based on the current state of knowledge, the application of such technologies must be viewed very critically. Regardless of whether this system would be additionally equipped with a gene drive or not, retrievability is just as difficult to imagine here as is controlling the spread. This methodology also has a high potential for abuse. Therefore, research in this area is absolutely necessary, but an active application

7 Karberg

S (2018) Crispr ist nicht immer Gentechnik. Der Tagesspiegel. https://www.tag esspiegel.de/wissen/europaeischer-gerichtshof-vor-der-entscheidung-crispr-ist-nicht-immergentechnik/20864058.html. Accessed: June 26, 2020. 8 Simon S, Otto M, Engelhard M (2018) Synthetic gene drive: between continuity and novelty: Crucial differences between gene drive and genetically modified organisms require an adapted risk assessment for their use. EMBO Reports 19:e45760–4. https://doi.org/10.15252/embr.201 845760. 9 Reeves RG, Voeneky S, Caetano-Anollés D et al. (2018) Agricultural research, or a new bioweapon system? Science 362:35–37. https://doi.org/10.1126/science.aat7664.

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in the field, according to the current state of knowledge, is associated with high risks and dangers. Which gene therapy approaches are available for humans? In September 1990, the first officially approved gene therapy was carried out in the USA. In September 2020, 3015 gene therapy studies were registered worldwide, 688 of which took place in Europe and 106 in Germany. Gene therapy refers to the introduction of new and the modification of existing DNA sequences in the genome for the treatment or prevention of diseases. Only gene therapy on body cells (somatic cells) is permitted. These are all cells that are not used for reproduction in humans. As early as 1985, a group of experts on “In Vitro Fertilization, Genome Analysis and Gene Therapy”, set up by the German Federal Ministries of Justice and of Research, came to the conclusion in its final report that the insertion of genetic material into somatic cells does not fundamentally differ in its ethical dimension from organ transplants. The German Medical Association (Bundesärztekammer) came to the same conclusion in its guidelines on gene therapy published in 1989. The situation is completely different with germ cells, i.e., egg and sperm cells, from which the embryo develops after fertilization. These so-called germ line cells are subject to the German Embryo Protection Act (Embryonenschutzgesetz), which also defines them very precisely as “all cells that lead in a cell line from the fertilized egg to the egg and sperm cells of the human being who has emerged from it, as well as the egg from the insertion or penetration of the sperm cell until the fertilization completed with nuclear fusion.” Furthermore, the law clearly states that “artificial modification of the genetic information of human germ line cells” is prohibited. Thus, in Germany, embryos may be created for the sole purpose of inducing pregnancy, for example, in the course of artificial insemination. Regardless of the German view on the handling of germ cells, germ line research is already being carried out in other countries. Scientists in the team of the UK developmental biologist Kathy Niakan at the Francis Crick Institute in London have been allowed to use the CRISPR/Cas system on human embryos since February 2016. Clearly, this research is not about researchers breeding babies in the laboratory. It is about basic research during early division of fertilized eggs. One of the research questions is the occurrence of side effects after genetic engineering treatment of the cells. In contrast to Germany, where the Embryo Protection Act clarifies that the “fertilized human egg cell capable of development from the time of nuclear fusion” represents life worthy of protection, in England, this only applies from the fourteenth day after fertilization. Therefore, in Niakan research, embryos are killed after seven days. The 14-day rule applies to embryo researchers in countries such as Australia, Canada, the USA, Denmark, Sweden

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and the UK. The deadline for using artificially created embryos for scientific purposes at the latest fourteen days after fertilization is based on developmental biology. Around the fourteenth day, the so-called primitive streak is created, which is the first sign of a developing nervous system. In addition, after the fourteenth day, it is impossible for the embryo to divide to form twins. Before that, the formation of identical twins would be possible, which is seen by some as a lack of individuality. In 1984, therefore, English scientists proposed that embryo research should be permitted until that date. Since 2016, when scientists succeeded in keeping human embryos alive outside the uterus for more than fourteentdays after in vitro fertilization, this rule has been hotly debated. The first reports of gene edited human embryos come from the USA and China. In May 2015, Chinese scientists from Sun Yat-sen University in Guangzhou/China reported that they had genetically modified human embryos using the CRISPR/Cas system.10 To be precise, they reported the problems, as most embryos died prematurely. Again, the scientists did not have the primary goal of creating genetically modified humans. They therefore worked with so-called tripronuclear zygotes that develop into triploid embryos. This means that the genetic material is present three times in all cells. As a result, these embryos die early. It is clear that human germ line intervention for research purposes is in full swing.11 Perhaps the most important ethical question in the near future will be how we want to deal with the knowledge and possibilities gained—and this against very different sociocultural backgrounds. If a serious hereditary disease cannot be treated but repaired, do not doctors have to help? How is abortion to be weighed up against gene therapy intervention in the germ line? These are only a few questions that need to be addressed.

10 Liang P, Xu Y, Zhang X et al. (2015) CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein & Cell 6:363–372. https://doi.org/10.1007/s13238-015-0153-5. 11 At the time of going to press, it became known that the Chinese scientist He Jiankui used the CRISPR/Cas system to gene edit embryos, which were then born as twins. More at: genegenome-gesellschaft.de.—This translated edition allows me to add the following paper for a compilation of the events: Greely HT (2019) CRISPR’d babies: human germline genome editing in the ‘He Jiankui affair’. J Law Biosci 6:111–183. https://doi.org/10.1093/jlb/lsz010.

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Writing the Genome

Whereas in the previous chapter, we asked very specific questions concerning gene therapy that affect humanity, it is now becoming a little more utopian—but only a little. The latest methods of genetic engineering involve not only the precise modification of the genetic material, but also the complete chemical synthesis of genetic information. The bacteria infesting virus (phage) FX174 was the first (by Frederik Sanger) sequenced genome in 1977—and a quarter of a century later one of the first completely synthetically produced genomes. A pioneer of genome research, Craig Venter, was involved in this work. The biochemist Eckard Wimmer, who is of German origin but works in the USA, had previously succeeded in synthetically generating an infectious poliovirus. Obviously, we are now able to “write” genetic information. This research is part of a new field of research: synthetic biology. Synthetic biology attracts attention with headlines like “Playing God” in popular magazines. They describe how scientists try to specifically modify microorganisms in order to give them functions such as the synthesis of biodiesel or the binding of atmospheric carbon dioxide.1 But this is essentially genetic engineering! In fact, genetic engineering has been dealing with similar questions for a long time. Synthetic biology, however, goes further in that it wants to elevate biotechnology or genetic engineering to a true art of engineering by working with standardized components, called DNA parts or BioBricks. As in the engineering sciences, the result of the recombination (synthesis) of such components should be predictable or simulable. Trial and error should be replaced by design. The biologist becomes a designer. As a modern subdiscipline of the biosciences, synthetic biology is currently at the forefront of a number of developments in genetic

1 Church

GM (2012) Regenesis. Basic Books, New York.

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engineering in recent decades.2 The term synthesis should be understood here in the sense of bringing together much like in chemical synthesis—DNA modules with defined functions are brought together and something new is created. The publication of the research results3 of the J. Craig Venter Institute in the USA on May 20, 2010, which was staged as a press conference and scientific publication, has had the greatest influence on the public perception of synthetic biology so far. It was announced that “the first self-replicating species that we have had on the planet whose parent is a computer […] the first species that has its own website encoded in its genetic code” has been created. The synthesis seems simple: “building the chromosomes from four bottles of chemicals.” So the first bacterium was created whose genome was chemically produced in the test tube. Scientists at the J. Craig Venter Institute have invested fifteen years of work and forty million US$ in the project. The resulting bacterium is called Mycoplasma mycoidesstrain JCVI-syn1.0. The international media response ranged from calm perception to hysterical Frankenstein reports. The Vatican took the news of the first synthetic species calmly and acknowledged the scientific achievement. This was not without a certain irony, as most press releases, at least in Europe, accused scientists of playing God. What was actually achieved by Craig Venter’s working group? First, a modified genome sequence of the bacterium M. mycoides was synthesized. The changes compared to the sequence template, which is about one million nucleotides long, mainly concern the integration of so-called watermarks, which make the synthetic genome clearly distinguishable from the original. For example, one watermark contains the URL to a website with information on the bacterium. The actual synthesis of the genome took place in several steps. First, the chemical synthesis of DNA fragments 1078 times 1080 nucleotides long was commissioned from DNA synthesis companies—including the company GeneArt from Regensburg/Germany. These were assembled in several steps in a lengthy process to form a circular chromosome 1,077,947 base pairs long. However, the fragment fusions were not carried out in vitro (in the test tube), but in vivo in yeast cells. Thus, the genome synthesis described is mainly based on biological functions of yeast. Only the initial synthesis of the short fragments was done chemically. Also the introduction of the produced M. mycoides genome into M. capricolum was carried out with a standard method from cell biology, the protoplast fusion. In sober terms, the scientific success of this research project may be 2 Synthetic biology is not synthetic evolutionary biology. The latter is an extension of Darwin’s

theory of evolution with modern scientific knowledge. DG, Glass JI, Lartigue C et al. (2010) Creation of a bacterial cell controlled by a chemically synthesized genome. Science 329:52–56. https://doi.org/10.1126/science.119 0719.

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reduced to the combination of old methods and the elimination of resulting new problems—as well as excellent press relations. Since both the yeast and the target protoplast had to be functional and completely present, there can be no question of a new synthesis of life. “What I cannot create, I cannot understand,” the American physicist and Nobel laureate Richard Feynman once said. It is only a small step from the mathematical modeling of metabolic pathways and the prediction of their behavior with a computer to validation or use in a living organism—if the methodology for the targeted biogenesis of the desired building blocks exists. This outlines an important field of research in synthetic biology: the design of a metabolic pathway or even an organism and its incarnation. The results of the modeling provide the target. This subdiscipline of synthetic biology is known as metabolic design. It is a direct continuation of genetic engineering. Examples are the biosynthesis of the malaria drug artemisinin or of biodiesel with specifically designed microorganisms. Why is this more than genetic engineering? The essential difference lies in the approach. All biological functional elements are available as so-called parts together with a detailed description in an electronic database and as a physical DNA sequence in a freezer. The scientist can now select and order suitable components based on that description. The DNA sequences of the parts have a strictly defined structure that allows several parts to be arranged in sequence to form a gene construct (module) using standard molecular biology methods—even robots can do this. This construct can then be used to transform a target organism. A further area of synthetic biology deals with this target organism, which is consequently referred to as the chassis. The aim is to design a chassis that interacts as little as possible with the gene constructs to be taken up. There are two opposing approaches to this. In the research area of minimal cells, starting from a bacterium with a genome that is as small as possible, the researchers investigate how many genes are dispensable. M. genitalium has the smallest known genome of a free-living bacterium with almost 500 coding genes. Studies indicate that 430 of these genes are essential. A cell with just these genes was presented to the public as MycoplasmaSyn3.0 in 20164 and a patent application was filed. In contrast to this top-down approach, in which genes are removed step by step starting from an intact cell, the bottom-up approach of protocell research stands in contrast. Based on fat-droplet-like artificial cells, an attempt is made to bring genetic and biochemical components together and to make them grow and multiply. This

4 Hutchison

CA, Chuang R-Y, Noskov VN et al. (2016) Design and synthesis of a minimal bacterial genome. Science 351:1414–1425. https://doi.org/10.1126/science.aad6253.

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research approach is closely related and interwoven with the search for suitable conditions under which the development of life might have taken place. Synthetic biology also includes the attempt to design microorganisms that act as biological sensors and provide a defined response to certain environmental signals. For example, there are experiments with bacteria that have been optimized to detect the explosive TNT and—in the presence of TNT—to trigger a reaction cascade in the bacterium. This could be designed in such a way that the bacteria emit light signals. The declared aim is to use such bacteria for the detection of decaying landmines. It would also be conceivable to detect mixtures of substances with an organism and also to indicate concentration levels. This would require complex circuits, another branch of synthetic biology. The ultimate goal is logical signal processing as known from computers. The foundations for this have already been laid. The metaphor of programming a cell is hereby raised to a new level, from the programming of the DNA code to the programming of information processing by genetic control circuits. A great success was already achieved in the year 2000 with the targeted development and simulation of a circuit on the computer and its implementation in a cell. The company Microsoft Research has already developed a programming language and environment specially tailored to the requirements of synthetic biology called Visual GEC, where GEC stands for genetic engineering of living cells.5 Regardless of whether you are working with a chassis or a complex target organism: Interactions between the basic functions of the chassis and the inserted modules must be prevented. The subdiscipline orthogonal systems explicitly deals with this problem. For example, by using an unnatural genetic code and an expression apparatus adapted to it, the synthetic transcription and translation apparatus can be separated from that of the target organism. Orthogonal systems are often also referred to in the context of biosafety. This branch of synthetic biology deals with the risks of using synthetic microorganisms and overlaps almost completely with safety research in genetic engineering. Overall, biosafety measures serve to protect employees, the population and the environment from dangerous organisms and biological agents. A greater danger, however, is posed by the criminal or terrorist misuse of the possibilities of synthetic biology—a topic that is dealt with in the area of biosecurity. In this context, the publication of the relatively simple synthesis of the

5 Pedersen

M, Phillips A (2009) Towards programming languages for genetic engineering of living cells. Journal of the Royal Society, Interface 6:S437–S450. https://doi.org/10.1098/rsif. 2008.0516.focus.

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poliovirus and the Spanish influenza virus6 has given cause for alarm. The latter caused at least twenty million deaths between 1918 and 1919. The synthesis of the two viruses took place against the background of understanding the molecular mechanisms of infection and the extremely high pathogenicity. The public accessibility of the genome sequences of extremely infectious and deadly viruses, such as the Ebola virus, combined with the possibility of ordering DNA molecules tailor-made as merchandise, shows the explosiveness of what may be the simplest subarea of synthetic biology, DNA synthesis. DNA synthesis companies have therefore agreed to always check orders for sequence similarities with known pathogens—but this cannot prevent criminals from ordering DNA synthesis equipment on, for example, eBay. Like any technology, synthetic biology involves opportunities and risks, the respective potential of which must be weighed up. It is absolutely necessary for progress in synthetic biology to be monitored and evaluated by international control bodies. Developments in a completely different field, the computer sciences, show how difficult this will be, especially in the civil sector. Here, too, high tech is being developed not only by industry, associations and governments, but also by civilians. There are, in the sense of informational self-determination, great applications that can deeply intervene in society and topple state governments. There is, however, also the dark side: namely cybercrime. Here, single individuals, some of them even too young to be legally responsible, can cause great damage. Controlling this is another challenge in the field of genetic engineering and synthetic biology (see also Chap. 7).

6 Tumpey TM (2005) Characterization of the Reconstructed 1918 Spanish Influenza Pandemic

Virus. Science 310:77–80. https://doi.org/10.1126/science.1119392.

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Marketing Genetic Information

As should have become clear from the previous sections, the cash till is ringing in full swing in the area of marketing genetic information and methods. Probably not everyone has noticed that when the first draft of the DNA sequence of the human genome was announced in 2000, in reality two versions were presented. The representatives of these two versions stood next to the then US President Bill Clinton in the White House on June 26: on one side the geneticist and entrepreneur (Celera company) Craig Venter, on the other the geneticist Francis Collins as representative of the international, publicly funded Human Genome Organisation (HUGO). HUGO was the largest research project worldwide at that time. It was a publicly funded alliance of over 1000 researchers from forty countries with the declared goal of deciphering the 3.2 billion nucleotides of the human genome by 2015 and making them publicly available. Venter left HUGO as early as 1992, founded a research institute and pursued his own sequencing activities parallel to the public project. Germany joined HUGO in 1995 and contributed almost sixty million bases to the human genome via the participating research institutions in Berlin, Braunschweig, Heidelberg and Jena. While HUGO’s DNA sequences are freely available, customers had to pay for the Celera data set. Later, Craig Venter also offered numerous genomic data sets for sale, such as completely sequenced ecosystems. Of great importance, however, was Clinton’s announcement in 2000 that the human genome was not patentable: This caused biotechnology stocks to crash. Are shorter DNA sequences allowed to be patented? A great deal of controversy was unleashed on this issue when the biotechnology company Myriad Genetics was granted European patent number EP699754 in 2001 for the DNA sequence of the BRCA1 gene and an associated genetic test. About 10–15% of all breast cancers are caused by mutations in the BRCA1 and BRCA2 genes. Myriad Genetics had a monopoly on the diagnosis of the gene variants due to the patenting, also in many other countries, and at times © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. Wünschiers, Genetic Engineering, Springer essentials, https://doi.org/10.1007/978-3-658-32403-2_5

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charged over 4000 US$ for this. In 2004, the European Patent Office decided that the company’s original application did not refer to a novelty but only to a DNA sequence, and revoked the patent. However, Myriad Genetics appealed and in 2008 was granted a modified patent covering tests for certain mutations, but not the gene itself. In 2013, the US patent was also rejected on the grounds that it is not enough to isolate sequences to patent them. However, against the background that big data has also reached the life sciences and is analyzing DNA sequence data from patients to an unimaginable extent (e.g., in the UK BioBank Project 1 ), it is clear that knowledge is power. Whenever genetic association studies reveal the connection between diseases and genes, variants of these genes (alleles) and epigenetic changes (see Chap. 6), then this information can be used to develop lucrative genetic tests to be marketed as a service. The focus must always be on the patient and not the patent. It is therefore imperative that industry agrees on clear rules with policies that serve the public good, so that knowledge acquired in industry also benefits the general public. However, the example of Golden Rice, a rice variety that contains increased amounts of provitamin A (beta-carotene) as a result of genetic engineering, shows that this is not easy either. The development was started in 1992 by the German biologist Ingo Potrykus and the cell biologist Peter Beyer and published in 2000. The declared aim is to combat the vitamin A deficiency that is prevalent in many developing and emerging countries. The Golden Rice, named after the golden color of rice seeds due to their high vitamin A content, is regarded by some as a showpiece project, while others see it as a kind of Trojan horse of plant genetic engineering. Although the rice has not yet established itself, mainly for political and ideological reasons, it was awarded the Patents for Humanity Award by the US Patent Office in 2015. This honors the release of patented technologies for global humanitarian applications. This award points to a special feature of the Golden Rice Project: it is intended to serve the common good and the seeds are distributed without royalties. It sounds like a dream, but in the dispute about genetic engineering in general and the form of development aid to be provided in particular, it became a nightmare for Potrykus and Beyer. Nevertheless, research is progressing and field trials are being carried out in the USA, Vietnam and the Philippines, funded by the private Bill and Melinda Gates Foundation. In general, the path from the idea of generating a genetically modified organism to an approved commercial product is a very long and costly one. It is therefore not surprising that the seed business with genetically modified plants, 1 Canela-Xandri O, Rawlik K, Tenesa A (2018) An atlas of genetic associations in UK Biobank.

Nature Genetics 50:1593–1599. https://doi.org/10.1038/s41588-018-0248-z.

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for example, is dominated by a few large corporations such as Bayer (which has swallowed Monsanto), Syngenta, DuPont Pioneer, BASF or Dow. Companies have to be able to afford a licensing procedure with the necessary preliminary tests and studies that have to be presented. For example, it must be demonstrated in the application that the GMO has no adverse effects on humans, animals or the environment. In the case of food or feed, analyses must show that the GMO food does not differ significantly from conventional reference products and does not contain allergens—a commercially available kiwi would not pass this test. In addition, post-market monitoring procedures must be provided to identify the engineered organism. The application is then forwarded to the European Food Safety Authority (EFSA) for examination. The EFSA may require the applicant to carry out further testing, which is often costly and lengthy. The national authorities of the Member States are involved in the procedure and can request data. The EU reference laboratory validates the methods proposed by the applicant for detecting and identifying the GMO in question. Finally, EFSA forwards an opinion to the EU Commission and the Member States and makes it available to the public. The Commission then submits a proposal for a decision to the Member States. A qualified majority is required for adoption. This is when 55% of the Member States (currently 14 out of 27) agree and 65% of the EU population is represented. At best, the procedure takes nine months, but usually it takes several years. Applicants need a capital buffer in order to survive the duration of the authorization procedure. It is understandable that corporations recoup this investment from farmers in the form of license fees. The situation is similar with GMO-free, conventional seeds. Here, too, a long time of breeding work goes into the seed and, in Germany, an approval procedure follows at the Federal Plant Variety Office (Bundessortenamt) and, if necessary, its European counterpart, the Community Plant Variety Office (CPVO). Here, too, the farmer has to pay licensing and reproduction fees, especially for hydrides. In hybrid breeding, suitable, cultivated inbred lines are crossed with each other. The offspring of the first generation of such a cross often have agronomically more valuable characteristics compared to the parent generation, such as stronger growth or larger fruits. This is called the heterosis effect. However, further breeding of the hybrid variety is not economical, since sowing the seeds of the first generation are segregating into the parent traits in the second generation—the profitable heterosis effect is lost. Instead, the farmer has to buy new seeds again. To counteract the growing privatization in the seed sector, an alliance of breeders and lawyers has developed an open licensing model for seeds.2 This is based on comparable models in the software sector and 2 Open

Source Seeds. https://www.opensourceseeds.org. Accessed: June 26, 2020.

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only permits use if the breeder undertakes to place further developments under the same licensing model and not to have them patented. Currently, nine free varieties are available: three tomato, three wheat, one maize, one potato and one pepper variety. It remains to be seen whether the concept will prevail, as the development costs for new varieties or breeds must at least be covered. In the context of discussions on the exploitation of farmers with patented, genetically modified genetic material, a clear distinction must be made between technology on the one hand and economy on the other. Science must assess and minimize the risk of a technology. However, it is a question of business ethics to assess the consequences of business models for people and the environment and to show feasible ways forward. This is also the result of the so-called Monsanto Tribunal, which took place in The Hague/Belgium in 2016/2017.3 In a legal opinion, five judges explained how Monsanto’s (now Bayer’s) practices violate human rights and lead to ecocide, among other things. The tribunal was not an officially recognized court, as there is currently no legal instrument that allows the prosecution of companies and their directors as responsible for crimes against human health or the integrity of the environment. But the tribunal show once again that the responsibility of companies goes beyond the mere sale of a product.

3 Internationales

Monsanto Tribunal. https://monsantotribunal.org. Accessed: June 26, 2020.

6

Genetics and the Environment: Epigenetics

Överkalix is a small, remote place in northern Sweden. It became better known in 2001 through a sensational study that describes how environmental factors can affect heredity. In the specific Överkalix study, medical statisticians examined the influence of diet on the health status of first- and second-generation offspring.1 The scientists benefited from the fact that crop yields, birth and death certificates and health data of the test persons were available. They investigated the living conditions, in particular the nutritional status of the birth cohorts 1890, 1905 and 1920, which was predicted on the basis of the crop yields, and the effects on subsequent generations. It was found that malnutrition of male ancestors between the ages of nine and twelve years had a positive effect on the life expectancy of second-generation offspring. More precisely: the probability of grandchildren dying of heart disease or diabetes decreases. Wow. How can this be possible? There are several known mechanisms, cytosine methylation being the most prominent. Chemically speaking, a methyl group (–CH3 ) can be reversibly attached to an individual cytosine (C) in the genome, when the cytosine is followed by a guanine (G), so-called CpG dinucleotides.2 Methylated CpG dinucleotides cause a change in the activity of genes. This epigenetic gene regulation is based on the fact that access is literally made more difficult for the molecular apparatus that reads the genetic information. Methylation is dynamic, meaning that methyl groups can be added to and removed from DNA. Now it makes a big difference

1 Bygren

LO, Kaati G, Edvinsson S (2001) Longevity determined by paternal ancestors’ nutrition during their slow growth period. Acta Biotheoretica 49:53–59. https://doi.org/10. 1038/sj.ejhg.5201832. 2 The P stands for phosphate and is therefore used to distinguish CG dinucleotides within a DNA strand from CG base pairing of a DNA double strand. © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. Wünschiers, Genetic Engineering, Springer essentials, https://doi.org/10.1007/978-3-658-32403-2_6

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whether this epigenetic regulation affects body cells or germ cells, i.e., sperm or egg cells. Many plants show epigenetic regulations in the body cells. Changes in CpG methylations in plant tissue cells can easily be passed on to offspring. Since plants have the possibility of vegetative reproduction, methylations of DNA can easily be passed on to offspring that are actually clones. It is therefore probably no coincidence that the inheritance of acquired traits was first described by botanists such as Jean-Baptiste Lamarck. The Soviet agricultural biologist Trofim Denisovich Lysenko turned this Lamarckian inheritance of traits acquired during lifetime into a dogma and rejected Darwinism. This thinking was politically abused by the state leadership of the Soviet Union by transferring it to animals and humans.3 However, the transfer of acquired traits, manifested as methylations on DNA, to offspring of animals and humans requires an epigenetic alteration of the genetic material in the sperm and egg cells and a transfer to the embryo. It is easy to imagine that an embryo is epigenetically imprinted during pregnancy. In the Överkalix case, however, the information was passed on to the next but one generation, the grandsons. And this is remarkable because it was previously assumed that the germ line was strictly separated from the body cells by the so-called Weissmann barrier. The German evolutionary biologist and physician August Weissmann proposed this strict separation over a hundred years ago. And indeed, it was later discovered that epigenetic markers are reset during germ cell formation and after fertilization, known as germ line reprogramming. However, there are exceptions that we know as maternal or paternal genetic imprinting. An underlying mechanism for bypassing reprogramming was elucidated in 2012.4 Since then, further mechanisms have been proposed for how epigenetic imprinting can act over several generations. Epigenetics is undoubtedly one of the most exciting areas of genetics, providing those who need it with a molecular biological basis for our responsibility over future generations.5 There is now a separate field of research, Nutritional Genomics, dedicated to the interaction of food and genomes. In addition, research into epigenetics is expected to provide answers to disease patterns that cannot currently be explained genetically. The elucidation of the epigenetic influence on the efficacy of drugs also plays a role in the context of personalized medicine. And 3 Graham

L (2016) Lysenko’s Ghost—Epigenetics and Russia. Harvard University Press, Cambridge, MA. 4 Nakamura T, Liu Y-J, Nakashima H et al. (2012) PGC7 binds histone H3K9me2 to protect against conversion of 5mC to 5hmC in early embryos. Nature 486:415–419. https://doi.org/ 10.1038/nature11093. 5 Kegel B (2015) Epigenetik. DuMont Buchverlag, Köln.

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gene editing (see Chap. 3) already provides methods for modifying epigenetic markers. Scientists agree that there are still many unknown mechanisms of action to be discovered. However, this also contains a warning for genetic engineering: epigenetic effects must be included in the risk analysis for all interventions. A current example, again from the food sector, will illustrate the importance of this topic. We live in a time in which awareness of healthy food is widespread. However, this not only leads to a preference for consuming wholesome food, but also to the supposed upgrading of food with additives. These functional foods propagated by the food industry are intended to protect against deficiency symptoms and to convey a healthy attitude to life to the consumer. In some countries, the artificial addition of vitamins and other nutrients to food is even required by law. For example, in Europe, baby food is supplemented with vitamins, and in the USA, Canada and many other countries, flour is mandatory fortified with folic acid (vitamin B9). In principle, folic acid is an essential component of the diet and is recommended to pregnant women in the form of tablets as a food supplement—and that is a good thing since it prevents spina bifida and other birth defects. In adults, folic acid deficiency has been linked to a number of diseases such as cancer and Alzheimer’s disease. People who carry a rare gene variant (allele) of an important enzyme in folic acid metabolism (methylenetetrahydrofolate reductase) are particularly affected. Carriers of this gene variant (about 0.2% of the US population) suffer considerably more frequently from diseases, which are otherwise only caused by folic acid deficiency. The disease is therefore prevented by administering increased amounts of folic acid in the diet. However, in 2005 a study warned that a constantly increased folic acid intake leads to a preferred selection of the defective gene variant in the population, since carriers of the defective allele no longer show symptoms.6 Individuals who are carriers of the defective gene variant are helped by the constantly increased folic acid intake. However, ultimately this has a negative effect on the entire population.

6 Lucock M, Yates ZE (2005) Folic acid—vitamin and panacea or genetic time bomb? Nature

Reviews Genetics 6:235–240. https://doi.org/10.1038/nrg1558.

7

Citizen Science

For a long time, science was something for people who acquired special knowledge and applied it to answer a wide variety of questions. Nonscientists could only participate passively at best, for example, by making computing power of the private computer available for projects like SETI@home (Search for ExtraTerrestrial Intelligence at home) or the search for a vaccine against Ebola viruses. Since December 2014, more than 72 thousand years of computing power have been donated to the latter project in this way. Similarly, owners of more than 3000 dogs contributed to the identification of the genetic basis for the bright blue eyes of Siberian huskies.1 This commitment is known as Citizen Science. However, there is also the possibility to become active in private scientific research. For example, the Argentinean hobby astronomer Victor Buso observed a supernova in September 2016 and has thus become a co-author of the renowned science magazine Nature. In the field of genetic engineering, a community of interested people who call themselves do-it-yourself (DIY) biologists has been developing for several years.2 In so-called live hack spaces (laboratories), DIY biologists try to address more or less everyday questions. This also includes genetic engineering experiments. In the USA, some institutions or interest groups can rent laboratories with full equipment at low cost. Others work in the hobby cellar or the garage that has been converted into a private laboratory, which is why it is often referred to as garage laboratories. This also refers to the time in the 1970s, when pioneers such as Bill Gates with Steve Ballmer founded Microsoft and 1 Deane-Coe

PE, Chu ET, Slavney A et al. (2018) Direct-to-consumer DNA testing of 6,000 dogs reveals 98.6-kb duplication associated with blue eyes and heterochromia in Siberian Huskies. PLoS Genetics 14:e1007648. https://doi.org/10.1371/journal.pgen.1007648. 2 Wohlsen M (2012) Biopunk: Solving Biotech’s Biggest Problems in Kitchens and Garages. Penguin, New York. © Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. Wünschiers, Genetic Engineering, Springer essentials, https://doi.org/10.1007/978-3-658-32403-2_7

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Steve Jobs with Steve Wozniak founded Apple and turned the computer into a personal computer. There is always something hidden in the nimbus of innovative garage science. And here is the problem when it comes to genetic engineering. On the one hand, it is to be welcomed that there are interested people who want to get involved with genetic engineering. The difficulty, on the other hand, lies in the fact that downloading a manual on how to genetically modify an organism cannot be compared to a baking recipe. An unsuccessful meal may frighten the guests, a failed genetic engineering experiment and pouring the leftovers down the drain may have serious consequences. It is at least a matter of estimating the risks and taking the appropriate responsible actions. In 2017, an experiment kit from ODIN in the USA caused a sensation in Germany. The science kit costs US$ 159 and contains all the components needed to genetically modify a bacterium using the CRISPR/Cas system (see Chap. 3). During the ten hours of experimenting, which are spread over two days because of the cultivation of the bacteria, one bacterium (the intestinal bacterium Escherichia coli, which has been very well studied for decades) is made resistant to the antibiotic streptomycin. The bacteria used are a safety strain that can neither colonize the human intestine nor are they viable outside the laboratory. The experiment itself is based on research results from 2013.3 In the USA, the experiment is not regulated and there are videos on YouTube where the experiment is carried out in the kitchen—thus the kitchen becomes a biohacking space. In Germany, this is not allowed, but requires a level 1 genetic engineering safety laboratory and a certified scientist. Otherwise, a fine of up to EUR 50,000, or even three years imprisonment if a GMO is released, can be imposed. That this is a good thing was demonstrated at the end of 2016 when the Bavarian State Office for Environment and Consumer Protection (Bayerisches Staatsministerium für Umwelt und Verbraucher) found that, in addition to the expected E. coli bacteria, pathogenic representatives were also growing. Obviously, the bacteria supplied were contaminated. As a result, the import of the experimental kit was regulated. The European Centre for Disease Prevention and Control (ECDC) carried out a risk assessment in May 2017 and concluded: “[…] the potential contribution of the contaminated kit to the increasing burden of antimicrobial resistance in the EU/EEA is marginal, and the associated public health risk is considered very low.” Caution is therefore required, but there is no risk. However, the incident has also shown something else: The Bavarian State Office has denied German DIY biologists access to the detailed results. As a reaction, they contacted the renowned European Molecular Biology Laboratory (EMBL) in 3 Jiang

W, Bikard D, Cox D et al. (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology 31:233–39. https://doi.org/10.1038/nbt.2508.

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Heidelberg/Germany, where they were able to describe the contamination using DNA sequencing in certified laboratories with knowledgeable scientists. It is this open dialogue between scientists and DIY biologists that is fortunately blowing fresh wind into the genetic engineering debate in particular and the openness of science in general.4 However, despite all openness in dealing with genetic engineering, it must be clearly defined where the limits are. For example, the 28-year-old Tristan Roberts from the USA, who tested HIV-positive, genetically treated himself on October 18, 2017 in a Facebook livestream.5 In front of the camera, he injected himself with an unapproved therapeutic from the now shutdown Singaporean company Ascendance Biomedical, which incorporates a DNA fragment into his body cells, whereupon they form an active substance. There are numerous similar cases and the motivation ranges from DIY AIDS therapy to the hope of eternal youth through the introduction of gene constructs that induce cells to produce corresponding hormones. The US Food and Drug Administration (FDA) officially warns against such “unauthorized gene therapies.”6

4 Editorial

(2017) Biohackers can boost trust in biology. Nature 552:291–91. https://doi.org/ 10.1038/d41586-017-08807-z. 5 News2Share (2017) Biohacker Self-Administers Attempt at Gene-Therapy HIV Cure. https://www.facebook.com/N2Sreports/videos/1557343577706859/. Accessed: June 26, 2020. 6 Smalley E (2018) FDA warns public of dangers of DIY gene therapy. Nature 36:119–120. https://doi.org/10.1038/ng.3869.

What You Learned From This essential

I hope that this essential will give you an insight into the current possibilities of genetic engineering. Fascination and concern, possibilities and abuse, humility before the living and responsibility stay close together. This essential is not intended to provide answers, but rather to motivate you to ask questions—I have some: • Reading the genetic material of living beings provides fascinating insights into the history of evolution, for example, of humans. It also makes it possible to determine genetic markers for diseases or, for example, cognitive traits that can be used in diagnostics. However, this knowledge is associated with a high level of responsibility when it comes for example to prenatal or preimplantation diagnostics or marketing. When does diagnostics become breeding through targeted selection? • Editing of genetic information, for example by means of CRISPR/Cas, can be used to specifically modify living organisms. The simplicity of the application offers opportunities, for example, in animal and plant breeding, and risks, for example, when using the gene drive. In addition, genome editing makes it possible to cure genetic diseases—for example, in the human embryo. Are we allowed to do that? • Writing unprecedented genetic information that is new to nature on a large scale is the subject of synthetic biology. This new branch of genetic engineering aims to revolutionize biology—and attracts laymen who wish to participate. How should the liberalization of biotechnology and genetic engineering be controlled? • The genetic material is part of a cell and the cell is part of an environment. The most recent extension of our understanding of heredity, i. e. epigenetics, calls

© Springer Fachmedien Wiesbaden GmbH, part of Springer Nature 2021 R. Wünschiers, Genetic Engineering, Springer essentials, https://doi.org/10.1007/978-3-658-32403-2

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What You Learned From This essential

into question some centuries-old knowledge—and provides a molecular basis for Lamarck’s principle of the inheritance of traits acquired during a person’s lifetime. Do we have to learn to rethink the interaction between living beings and the environment?

Literature

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Related Literature Carroll SB (2005) Endless forms most beautiful: the new science of evo devo and the making of the animal kingdom. Norton, New York Diekämper J, Fangerau H, Fehse B et al (eds) (2018) Vierter Gentechnologiebericht. Nomos, Baden-Baden Henderson M (2010) 50 Schlüsselideen Genetik. Spektrum Akademischer, Heidelberg Mukherjee S (2017) The gene: an intimate history. Simon & Schuster, New York Schmid RD (2016) Taschenatlas der Biotechnologie und Gentechnik. Wiley, Weinheim

Relevant Fiction Bacon F (2016) The new atlantis. Createspace independent publishing platform (originally from 1624) Ishiguro K (2005) Never let me go. Faber and Faber, London Suter M (2017) Elefant. Diogenes, Zürich

Related essentials Ableitner O (2014) Einführung in die Molekularbiologie. Springer Fachmedien, Wiesbaden Gramer G, Hoffmann G, Nennstiel-Ratzel U (2015) Das erweiterte Neugeborenenscreening. Springer, Wiesbaden Heberer B (2015) Grüne Gentechnik. Springer Spektrum, Wiesbaden van der Ven K, Pohlmann M, Hößle C (2017) Social freezing. Springer Fachmedien, Wiesbaden

Recent Films and Series Biohackers (2020) created by Christian Ditter, Germany. Claussen+Putz Filmproduktion for Netflix. IMDb: tt9849210 Human Nature (2019) directed by Adam Bolt, USA. News and Guts Films. IMDb: tt9612680 Okja (2017) directed by Bong Joon-ho, South Korea/USA. Plan B Entertainment for Netflix. IMDb: tt3967856