Genome engineering has a rich history which, when outlined in order, shows an industry determined to aid humanity through the study of genetics. From conceptualizing the double helix to CRISPR edits, this industry has demonstrated a commitment to game-changing discoveries, all while adhering to a careful set of ethics and regulations.
There is a rich history of gene editing which, when outlined in order, shows an industry determined to aid humanity through the study of genetics.
Understanding the genome editing history is incredibly important to understanding the current state of the field. Some important events include the discovery of the double helix, recombinant DNA (rDNA), human cancer therapies, the invention of CRISPR, and more. In this guide, we have created a detailed timeline broken down into decades, with some of the most prolific discoveries and events in the history of genetic modification to its present state.
Let's start this brief history of genetic engineering with the earliest discoveries impacting the future of the field.
There were two main events that took place prior to the creation of recombinant DNA (rDNA), and the other prominent discoveries of the 1960s that put genome engineering on its path to revolutionize biology. The discoveries of the 1950s in the field of genetics paved the way for future study of genetics, biotech, and all things DNA-related.
The twisted-ladder structure of deoxyribonucleic acid (DNA) familiar to us today as the "double helix" was pioneered in 1953 by James Watson and Francis Crick, giving rise to the modern study of biology, and genetics. This was one of the most important early milestones that defined genetics, as we know it today, and was the backbone of many of the future discoveries that come out of the world of biology.
This discovery is widely considered one of the most significant early events in the field of genetics, yet an important person is often missing in the narrative. Watson & Crick owed their discovery to the work of their colleague Rosalind Franklin, without whose X-ray diffraction images of DNA proteins in the early 1950s, they never would have thought to consider the idea of the double helix.
Arthur Kornberg had been working on the project of DNA synthesis from about early 1950s. In 1953, when all five nucleotides could be synthesized in the lab, he decided to turn his focus to the remaining factors needed for DNA synthesis - the enzymes that assemble nucleotides into DNA or RNA. He isolated DNA polymerase from bacterial extracts and within a year successfully synthesized DNA in vitro for the first time. Kornberg was awarded the Nobel Prize for this outstanding achievement.
Interestingly, his initial papers publishing his DNA synthesis data were rejected by the The Journal of Biological Chemistry; some reviewers refused to term the product "DNA," and suggested the term "polydeoxyribonucleotide" (one demanded proof of genetic activity). Kornberg withdrew his papers until a new editor joined the JBC in 1958.
Important to the history of gene editing are the origins of genetic engineering that bring us back to Silicon Valley in the early 1960s. The '60s saw an explosion of research into the structure and function of prokaryotic and viral genetic material.
The green fluorescent protein (GFP) is naturally present in the Aequorea Victoria jellyfish and fluoresces with a green light when exposed to blue wavelength. In 1962, Osamu Shimomura isolated this protein, and researchers Martin Chalfie and Roger Tsien further developed it into an indispensable biological tool.
This accomplishment was incredibly important to the field of genetics because by fusing the GFP gene with another gene that produces a protein of interest in a plasmid, scientists can determine which cell expresses their target gene. In 2008, the three researchers together won the Nobel Prize in Chemistry for their discovery and development of the green fluorescent protein.
The discovery of DNA ligases is considered a pivotal point in molecular biology, because they are essential for the repair and replication of DNA in all organisms. Essentially, catalyzing the formation of a phosphodiester bond allows for DNA strands to join together. This helped pave the way for other "splicing" experiments in the 1960s and early 1970s, a combined effort by Gellert, Lehman, Richardson, and Hurwitz laboratories, which led to the creation of recombinant DNA.
This idea of restriction enzymes started as a hypothesis by Werner Arber who noticed that certain bacterial strains fought off bacteriophage infection by chopping off its DNA. Why do these molecular scissors not cut off the bacterium's own DNA?
Arber hypothesized that bacterial cells produce two types of enzymes: one called a "restriction" enzyme that can identify and cut foreign DNA, and a "modification" enzyme that recognizes the host DNA and protects it from cleavage. This hypothesis was proven in an experiment in which two enzymes were isolated from E. coli. The modification enzyme, methylase, protected DNA of the bacterium, while the restriction enzyme chopped off phage (non-methylated) DNA.
By 1972, researchers had created the first chimeric recombinant DNA cloning SV40 molecule into plasmid DNA. At this point in the history of gene editing, this decade demonstrates the fundamental achievements that sculpted genetics for all future scientists.
Hamilton Smith, a molecular biologist at Johns Hopkins University School of Medicine, had been working on the bacterium Haemophilus influenzae Rd in the 1970s. In 1972, he successfully purified the first site-specific Type II restriction enzyme called Hind II from this bacterium. He and his team also identified the 6 base pair phage DNA sequence that Hind II recognized for the site-specific cleavage.
This understanding of how restriction enzymes "cut" DNA, and how host DNA works to protect itself is the basis for the modern genetic engineering therapies that are being developed, and for CRISPR.
Paul Berg became the first scientist to ever accomplish creating recombinant DNA from more than one species, which came to be known as the "cut-and-splice" method. He cut the DNA from two viruses creating "sticky ends;" the DNA was then incubated, the ends would anneal on their own, and the addition of DNA ligase would seal it.
This proved the validity of the theory that it was possible for any two DNA molecules to be covalently joined together. This achievement was considered a fundamental step in the field of genetic engineering, and was the biggest stepping stone toward the creation of recombinant DNA.
Paul Berg was awarded the Nobel Prize in Chemistry in 1980 (shared with Walter Gilbert and Frederick Sanger) for "his fundamental studies of the biochemistry of nucleic acids, with particular regard to recombinant DNA."
Once the first restriction enzymes were discovered, Daniel Nathans tested them on SV40 viral genome. He found that the restriction enzyme that Smith discovered cut the viral genomic DNA into 11 fragments. Extending their work with discovery of more restriction enzymes in the future, Nathans demonstrated the utility of restriction enzymes in mapping out DNA sequences.
There were also able to separate fragments of viral DNA using gel electrophoresis. This leap made by Nathans helped take Smith's discovery to the next level, and was key future work in mapping out the genome of many different organisms.
In 1978, Werner, Nathans, and Smith were awarded the Nobel Prize in Physiology or Medicine for discovering and isolating restriction enzymes, and recognizing their applications to solving the problems of molecular genetics.
These experiments and discoveries with DNA were all leading to this moment: the creation of recombinant DNA (rDNA). This rDNA, essentially, is DNA that has been created through the combination of elements of DNA from different organisms. By introducing genetic material from one organism to another, this discovery established the principles of modern genetics, and was the basis of many future experiments.
The key to rDNA is that it can replicate naturally, despite being artificially introduced in another organism, and this was the achievement of Stanley N. Cohen & Herbert W. Boyer in 1972.
All these developments were great for science, but with these recent achievements in splicing DNA together, it was evident to The National Academy of Sciences that there were ethical dangers associated with these experiments. They proposed a temporary moratorium on all genetic engineering experiments in 1974.
Seeing the importance of this field of study, Paul Berg, the recombinant DNA pioneer, organized the Asilomar Conference in February 1975, which was attended by over 100 scientists in the field. At this conference, many of the ethical ideas surrounding genetic experiments were developed and agreed upon, and are still adhered to to date with modern genetic engineering.
Joshua Lederberg, a Stanford professor, emerged from this conference as a strong voice highlighting the potential of recombinant DNA technology for curing disease. His optimism and foresight drowned out fears about using this technology for ill, including eugenics or "superbug" infectious diseases. Now a nobel laureate, Lederberg's argument for the successful future use of recombinant technology ushered in a golden age for science and biotechnology.
A human B cell has the amazing natural ability to clone itself, producing identical antibodies known as "monoclonal antibodies," which help fight immune diseases. George Kohler & César Milstein found a way to mimic this effect by merging myeloma cells (a cancer of plasma cells) with B cells producing antibodies. This fusion makes the cell divide rapidly, allowing for large numbers of antibody-producing cells to be grown.
Interesting, their work began independently, as Kohler had created cells that produce very specific antibodies, but didn't survive for long periods of time, while Milstein had antibodies with unknown specificity, but that multiplied and grew forever. By combining their work, they were able to make monoclonal antibodies with amazing precision, that divided and continued to do so - and survive - forever, essentially revolutionizing modern genetic diagnostics and immunology treatments.
The 1980s was a point in the genetic engineering timeline that turned many genetic experiments into real-world products and ideas, changing the landscape of what was to come in the 1990s. Focussing on vaccines and treatments, the 80s was a period that clearly displayed science's dedication to solving many human problems using our own DNA.
A transgenic animal is any animal that has had a gene from a foreign organism deliberately inserted in its genome. Today, generating transgenic animals for scientific experiments is quite common using modern genome editing methods, but as for the history of genetically modified animals, this process first began with a research team led by Thomas Wagner at Ohio University in 1981. Wagner and his team transferred the gene of a rabbit into the mouse genome by using a method now standard in genetics known as "DNA microinjection." Their experiment paved the way for the revolutionary transgenic animal experiments in the years to follow.
A company in the biotech game for over 40 years, Genentech scientist, Dennis Kleid, helped put the world's first genetically engineered drug - insulin- on the market for humans. Insulin had been used for the better part of a century to treat patients suffering from Type I diabetes. Historically, insulin had been harvested from animals, and while similar to human insulin, animal insulin has a few significant differences.
Moreover, in 1978, a single pound of insulin required 8000 pounds of pancreas glands from 23,500 animals. This required 56 million animals per year to meet increasing demand in the United States; a synthetic alternative was clearly necessary. This is considered a defining moment of Genentech's history, as well as a defining moment for the concept of genetically engineered drugs being approved for human consumption.
The discovery of the polymerase chain reaction (PCR) was integral in later DNA experiments and breakthroughs, as it is used to make many copies of a specific DNA segment. By implementing the chain reaction, any copy of a DNA sequence is amplified to make more copies, and can generate thousands or even millions of copies.
This discovery of the PCR technique by Kary Mullis made DNA far more accessible to scientists by reducing the time it takes to "clone" DNA, which previously required cutting it into pieces of thousands of base pairs, isolating them and growing them in bacterial colonies, finding the right segment, and then growing it further. The factor by which this technique made DNA experiments more efficient and "doable" cannot be understated.
Possibly the first technique for highly targeted genome engineering, the discovery of zinc finger nucleases (ZFN) improved the effectiveness of gene targeting in several ways. Zinc fingers help "recognize" DNA, making it possible to design ZFNs for a variety of different genomic targets varying throughout many different types of cells and organisms.
Each ZFN binds specifically to a set of three base pairs, and can be combined to recognize longer DNA sequences. Researchers have fused the DNA cutting enzyme FokI to the ZFN DNA binding domain to create "genomic scissors" which can cleave DNA at a specified location, creating a double stranded break (DSB) in the DNA. If a repair template is co-transfected into the cell, then a fraction of the cells will undergo homologous recombination, where the cell incorporates the template of interest into the specific breakpoint.
This new technique helped promote the idea of "backward" genetics over "forward" genetics: with forward, the scientist generates random mutations looking for something significant, and then characterizes the gene they believe is responsible for the mutation. With backward genetics, thanks to gene isolation methods like ZFNs, we are able to identify the exact gene causing mutation, and attack it specifically.
Around 1986 (and a couple of years prior), scientists were working on developing a vaccine for Hepatitis B. Previously in 1963, Baruch Blumberg developed a blood-derived vaccine for Hepatitis, which was approved for market in 1981. Once Pablo D. T. Valenzuela was able to create the world's first recombinant vaccine using yeast cells, primarily Saccharomyces cerevisiae.
This vaccine immediately became the market standard, and the blood-derived vaccine was removed from circulation. By establishing a recombinant process for vaccines, many vaccines we still use today were created, including for diseases such as: HPV, whooping cough, pneumococcal, meningococcal, Haemophilus influenzae type b (Hib), and shingles.
Following the 1980s trend of putting gene-altered organisms on the market, 1988 was the first time that a GMO crop actually appeared in fields in the United States (officially), and that crop was corn. Known as "Bt corn" because it contains genes from the bacterium Bacillus thuringiensis (Bt), this corn was able to increase yields by discouraging pest impacts.
Though herbicide-tolerant plants were also a subject of intrigue at the time, it was transgenic pest-protected plants like Bt corn that hit the fields first, and in this case, with a resistance to the tobacco mosaic virus (TMV). The first field trials of a tomato resistant to the same virus began this year, but would not hit markets until 1994.
From 1990 to 2003, the Human Genome project succeeded in mapping the human genome with more than 20 thousand genes identified and their genomic loci documented. In 2003, the ENCODE (Encyclopedia of DNA Elements) project kicked off, with the aim of creating a complete list of the functional elements of the human genome, including elements that act at both the protein and the RNA level, including regulatory elements controlling transcription, translation and replication. The 1990s was a period of discovery, and proving that the work being done in this field was not only valid, but necessary.
Many people regard CRISPR to have been pioneered by Jennifer Doudna & Emmanuelle Charpentier, however, the discovery of the principle of CRISPR (Clustered regularly interspaced palindromic repeats) was discovered by Francisco Mojica during his work with bacteria in the marshes of Santa Pola, when he noticed that parts of the DNA in the bacteria repeated many times, with regular spaces in between.
Over the next 10 years, Mojica continued to look deeper into these repeats until his critical discovery in 2003 that the repeating DNA matched alongside pieces of DNA that matched the viruses attacking the bacteria. This principle led to many other advancements in DNA study over the next decade that brought the tool of CRISPR gene editing to the forefront of today's study of genetics.
This tomato was approved at the same time as Bt corn, and was actually brought to market for public consumption in 1994. Scientists at Calgene believed that the polygalacturonase enzyme in tomatoes caused the dissolving of cell wall pectin, and by introducing a "reverse" copy of the gene, they could effectively reverse this process.
Because of their product's ability not to ripen, they named it, the "FLAVR SAVR." Unfortunately, it did not meet the anticipated level of success, which at first was paramount, but ultimately, resulted in commercial collapse. This product was a perfect example of how difficult it can be to bring genetically engineered products to market - especially crops - as people literally have to eat them, and therefore, are more weary of possible side effects.
This was one of the biggest stories spread through the public that science has ever seen, as "Dolly" the sheep was cloned in 1996. The project was led by Ian Wilmut of the Roslin Institute. As the first mammal to be cloned from an adult cell, with the same genetic identity, Dolly was a huge achievement, proving that the process of cloning found in nature could be attributed to organisms it does not naturally occur in.
Later in 2003, the first cloning of an endangered animal took place with the banteng, which was a great example of how cloning could help save species of animals that may not still be here in our lifetime.
In 1988, the United States Congress funded The Human Genome Project, which aimed to completely map out the human genome. Though completion of this endeavour wasn't until 2003, in 1999, scientists working on the project demonstrated that they had completely mapped out the sequence for chromosome 22.
This was a milestone for the project, and proved that the massive time and worldwide collaborative effort that went into this project wasn't for naught. This publication also confirmed the efficacy of the current sequencing methods in use by genetic scientists, and provided invaluable insight into the connection to certain diseases through human DNA.
Presently, thousands of other organisms' genomes have been sequenced and annotated, and technology is such that on the order of millions of human genomes have been sequenced to the tune of a couple hundred dollars a genome. Resources such as the UC Santa Cruz Genome Browser and Ensembl were developed which allow the genome and its known elements to be easily explored electronically.
Further developments in the history of genetic manipulation are seen by the progress in the mapping of the human genome at the end of the 1990s, which was a great way to cap off a decade that showed incredible promise for the years to come. New discoveries had shows that methods being used truly worked, which opened the door for brand new experiments.
An amazing development in the history of gene therapy and a great way to start out the 2000s, a well-conceived targeted gene therapy was approved by the U.S. FDA, and was sold as an anticancer drug to treat chronic myelogenous leukaemia (CML). This drug was called Glivec (imatinib), and is still used today as a cancer treatment drug.
Taking a more fun approach to the idea of genome editing, Alan Blake and Richard Crockett developed the first animal for sale as a pet that had been genetically altered. The "Glo-Fish" - literally a fish that glows - uses the naturally-occurring fluorescence in fish to create sub generations of fish that also glow.
While more of a fad than a lasting scientific achievement (as more significant things have been done with the fluorescent protein in fish), this marks the start of an era in which the public was more open to the idea of consuming gene-altered organisms.
In 2004, the United Nations formally endorsed biotech crops as a way of supporting struggling farmers in developing nations, and solving the world's hunger crisis.
In 2006, there was an on-trend breakthrough with using gene-editing to find cancer treatments, and that breakthrough was Gardasil. This was the first preventative cancer vaccine to ever reach the market. At this time, it was approved only for females aged 9-26, though in 2009, it was also approved for males 9-26. The popularity of the vaccine was substantial, considering the fact that it is a recombinant vaccine.
By 2018, Gardasil was approved for males and females aged 9-45, showing its effectiveness in the decade it had been on the market, and through continued study. To date, this HPV vaccine is still the only preventative cancer vaccine, though the Hepatitis B vaccine is known to reduce the risk of liver cancer.
Another immensely popular discovery in this period was in the field of stem cell research. Embryonic stem (ES) cells maintain pluripotency i.e. ability to differentiate into any cell type. However, using stem cells for developing therapies involved relying on isolation of cells from IVF embryos (unused ones).
In 2006, Dr. Shinya Yamanaka from UCSF first introduced the induced pluripotent stem cell (iPSC) technology. His team isolated fibroblasts (first from mice, later from humans) and reprogrammed them into stem cell state via expression of a mix of four genetic factors. These iPSCs could then differentiate into any specific cell type that the researchers wished to investigate.
In the present decade, the development therapies that have been theorized and studied for years are finally being approved for humans, as the discovery of CRISPR, possibly the greatest achievement in the history of genetics, takes hold on the world.
Breaking into the next decade, we see a scientist creating the world's first synthetic life form, which technically means a living organism that was entirely built rather than evolved or born. In this experiment, a 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome was created by Craig Venter and his team, and transplanted into a M. capricolum recipient cell to create the new M. mycoides cells controlled entirely by the synthetic chromosome.
This marks an important stage in the field of biology, as it proves that life can be created synthetically, which has a great number of applications in the world of genetics.
While ZFNs represented a revolutionary change in that they were able to manipulate the genome, their DNA binding domain is notoriously hard to design for efficient cutting at the site of interest. The design process, therefore, can take months and still yield unsatisfactory results with off-target effects.
In 2011, generation two of designer nucleases came in the form of transcription activator-like effector nucleases, or TALENs, which recognize a single nucleotide rather than a trinucleotide motif. Compared to ZFNs, TALENs benefit from ease of design. Effective TALENs can be designed and manufactured in a few days, and can be multiplexed on the order of hundreds at a time.
TALENs appear to have fewer constraints on genome site, and can be easily targeted across the genome. TALENs were also found to have less off-target effects than ZFNs, and are less cytotoxic for the host cell. While TALENs have clear advantages over ZFNs, they also exhibit a few disadvantageous properties. They are significantly larger than ZFNs, and therefore can be harder to deliver and express in vivo. Along these lines, while knocking out a gene with ZFNs and TALENs proves fairly straightforward, knocking in a gene with this technology is more complicated.
In 2012, Jennifer Doudna , Emmanuelle Charpentier, and their teams elucidated the biochemical mechanism of CRISPR technology. By making precise targeted cuts in DNA, CRISPR ushered in endless potential in areas of medicine, agriculture, biomaterials, etc.
CRISPR-Cas9 is a bacterial adaptive immune system in nature, whereby pieces of DNA from invading viruses are snipped off by a bacteria nuclease, CRISPR associated protein. The DNA fragment that is chopped off is saved as memory for fighting future infections. The CRISPR-Cas9 system can be engineered to edit eukaryotic DNA by designing guide RNA complementary to the target sequence.
The guide RNA has a 20 base pair protospacer motif with flanking homology to the cut site of interest. Cas9 binds to this protospacer motif in the guide RNA, which in turn binds to the site of interest. Cas9 then binds to a protospacer adjacent motif (PAM) in the genomic DNA, and catalyzes a DSB in the DNA at a position three base pairs upstream of the PAM. If a homology arm is provided with the CRISPR-Cas9 cassette, HDR will occur, otherwise the cell will employ NHEJ to create small indels at the cut site of interest.
What CRISPR technology is being used for varies from developing cancer treatments, to tackling obesity, to creating hornless cows - and so much more. Whether it's diagnostics, treatment, or something else, there are new discoveries every day coming to the forefront of this field.
Just as significant was Feng Zhang's work in demonstrating the utility of CRISPR outside of the world of bacteria- in genetic manipulation of eukaryotic cells. His lab aims to use CRISPR to understand the functions of the brain using optogenetics, in which light is used to control genetically modified neurons to develop therapies that will treat brain disorders.
Though CRISPR was seen as an incredibly revolutionary potential treatment for countless diseases, Kevin Esvelt took the idea to a whole new level when he realized the potential of CRISPR gene drives. Gene drives are genetic elements that "cheat evolution" by biasing their transmission to off springs beyond Mendelian laws. CRISPR based gene drives have promising potential in eradication of malaria and other vector borne diseases, as well in saving endangered species.
Esvelt realizes the potential danger of a system like this; the implications within even a small ecosystem could be catastrophic, and it would take immense amounts of testing before something like this was ever released. That's why the current studies of gene drives take significant precautions.
The salmon produced by the company AquaBounty were the first to be sold for human consumption as a genetically engineered animal. This process was more than 25 years in the making, and approved because of the simple need to provide a solution to the world's overfishing crisis.
In 2015, CRISPR was used to edit human embryos by Junjiu Huang at the Sun Yat-Sen University in Guangzhou. Originally rejected by Western science journals because it did not follow ethical rules of science, this later made its way into publication in other ways. Because Huang had altered the germline cells that affect heredity, his experiment aiming to fix a gene error causing a blood disease was not considered ethical, and became a controversy almost instantly. This was three years before human trials for CRISPR were officially approved by any governing body.
In 2017, two CAR T-cell therapies were approved: one for acute lymphoblastic leukemia in children, and one for advanced lymphoma in adults. This is a huge step for CAR T therapy, which, if treatments prove as effective as expected, will likely replace chemotherapy as the primary form of cancer treatment, as CAR T is not toxic to the human body, and has demonstrated complete ablation of cancerous tumors in as little as 10 days.
In a joint initiative by two companies, Vertex Pharmaceuticals & CRISPR Therapeutics, an experimental treatment for the blood disorder beta-thalassemia has been approved to start clinical trials. If successful, this therapy could end cases of this disorder, and sickle cell anemia. It would pave the way for future CRISPR treatments, changing the way disease is treated for humans.
In October 2019, Andrew Anzalone, a postdoctoral fellow from Dr. David Liu's lab, along with his colleagues published a paper on the development and application of prime editing in Nature. This novel gene editing technique can perform targeted small insertions, deletions, and base swapping in a precise way, while limiting negative effects. What sets this tool apart from CRISPR is its ability to do targeted editing without making double stranded breaks.
In the summer of 2020, the results of the CRISPR clinical trials began to slowly trickle in. Victoria Gray was the first patient to undergo the sickle cell disease treatment and her promising results started making headlines. Less than six months later, at the ASH meeting that took place in December 2020, the data presented showed that a total of ten patients that had received the CTX001 therapy had made significant progress. Seven of these patients were being treated for beta-thalassemia and the remaining three for sickle cell anemia. All of these patients showed a great improvement in fetal hemoglobin levels in their blood, had relief from bouts of pain and no longer required blood transfusions.
CRISPR also made headlines in October 2020 when it was announced that Emmanuelle Charpentier and Jennifer Doudna finally won the Nobel prize in chemistry for the development of CRISPR.
We hope you enjoyed this history of genetic engineering timeline, but the journey of genome engineering is far from finished. With discoveries burgeoning across the globe, human health and disease, along with areas such as agriculture, energy, animal husbandry, and environmental science among others all stand to benefit enormously from these strides forward. As CRISPR becomes as easy a tool to implement as hammer to nail, the world as we know it is continually bettered by the efforts of scientists who drive genome engineering technology forward.
If you're interested in the history, you're probably also interested in what's going on now! If you're looking for updates, be sure to check out our huge list of the best CRISPR news sources. If you have ideas of your own to share, or a story you think our readers would love, be sure to contact us because we would love to get your feedback.
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