Bio- and Gene Technology

“Biologists create artificial life”, “May man play God?” These and similar headlines can be found increasingly in the daily press, especially in the last few years. However, the research that is actually behind the articles is often difficult or impossible to understand for most readers. What is CRISPR and what does it mean when it is said that you can “edit genomes” with it?

In my article What is a gene? I have sketched a story that slowly approximates the nature of genes, i.e. the units of genetic information. And I have described how in the late 1960s, with the deciphering of the genetic code, essentially all basic principles of information storage in cells were clarified. It was now a matter of applying this knowledge and intervening in the genetic make-up of bacteria, plants or animals. On the one hand, manipulations of this kind promised further insight into the basic principles of all living cells in even greater scope and detail. On the other hand, it offered the perspective of influencing biological systems in a way that they would perform useful tasks for humans which they are naturally not capable of.

For these purposes, Paul Berg created (if you want to call it that) the first so-called recombinant DNA in 1972: he combined DNA fragments from different organisms and thereby obtained a DNA strand that would never be found in this form in nature and that contained properties of both starting fragments. All of this was made possible by the recently discovered restriction enzymes that were discovered in bacteria. These enzymes protect bacteria from viruses by cutting their DNA at certain points. These places are always the same for a particular enzyme; the restriction enzyme EcoRV isolated from Escherichia Coli, for example, cuts every DNA at all points where the sequence sequence GATATC occurs. In a human genome, this sequence occurs about half a million times. A complete genome that is digested with such an enzyme is then available in fragments averaging around 5000 base pairs. And if you should identify fragments on which a certain sequence lies, which you might want to use elsewhere, then you could do that, because the pieces of DNA can also be recombined again. Even pieces that came from very different organisms can be glued together this way.

The enormous potential of this technology was immediately evident in the research area, which led to the fact that leading representatives of this field came together only two years later, 1975 in Asilomar, for a historical conference to impose limits, rules and prohibitions on their own research. Less than two years later, in 1976, the first company was founded that wanted to use this new technology commercially. The company’s success proved it to be right; it was soon possible to use bacteria, into which human genes were introduced, to produce a number of clinically highly relevant substances. The human gene for insulin, for example, was introduced into bacterial cells, which reduced production costs immensely. Growth or coagulation factors were also soon produced with the help of recombinant bacterial cells.

In the following 1980s, this sector boomed. The first transgenic mice were generated for research and first field tests were carried out with transgenic plants. At first, a lot of it happens without much public interest. However, at the latest since 1996 the clone sheep Dolly was described and the race to decipher the human genome had begun, public interest in these developments has also increased. At the same time, however, things became more complex, so that hardly anyone without a degree in biology was able to make a qualified risk assessment. Many people felt overwhelmed by the advances in modern biology and the rate at which this field was developing did not seem to decline.

The latest headline-making technique is CRISPR. And because it is no exaggeration to say that CRISPR is revolutionizing biomedicine, this technology will be described here with a little more scrutiny.

CRISPR / Cas9 – Genome Editing for Everyone?

In 2012 an article roused the scientific world. Scientists from two teams, one led by Jennifer Doudna in California and one by Emmanuelle Charpentier in Basel, published an article in Science magazine. It described the application of an immune defense mechanism from bacteria, which was only discovered a few years earlier, for the purpose of targeted alteration of genetic information. The rather bulky name Clustered regularly-interspaced short palindromic repeats is consoled by the more than catchy abbreviation CRISPR. Since then, CRISPR has perhaps had an unprecedented success story; almost every biomedical researcher responded instantly.

The restriction enzymes that were discovered in the 1970s and that can cut DNA at certain points made it possible to produce recombinant DNA. However, a certain enzyme always cut at a certain point that cannot be influenced. In addition, the sequences that are recognized and cut are quite short and therefore occur relatively frequently in a genome.

The CRISPR system consists of a protein called Cas9 and a short non-coding RNA. The protein forms a complex with the RNA and scans the genome for a sequence that is exactly complementary to the sequence on the short RNA. By offering the protein an RNA whose sequence you have chosen yourself, you can send the protein to any location in the genome. This is exactly where the CRISPR/Cas9 complex cuts both strands of the DNA helix.

Image from Mariuswalter for Used under CC BY-SA 4.0 License. Many Thanks.

A DNA double-strand break is a catastrophic event for a cell. In order to survive, two different repair mechanisms can be activated: non-homologous end joining (NHEJ) or homology directed repair (HDR).

In NHEJ, the cell simply tries to recognize any open DNA ends and sticks them back together. However, errors often occur here, which means that a few base pairs might be lost or added at the break point. If this takes place directly within a gene, the reading frame can shift, so that the gene no longer makes sense, i.e. no corresponding functioning protein can be built. Thus, CRISPR is wonderfully suitable for switching off specific genes in a very targeted manner, which is of enormous importance for research. Until then, in some research models, such as the lovely zebrafish, it was not possible to switch off a certain specific gene in order to investigate the consequences of this loss of function.

But there is more. In the course of the second repair mechanism, the HDR, the cell tries to find out which sequence was on the DNA strand where a hole now gapes after the CRISPR cut. For this purpose, DNA strands, which carry the same sequence as the cut strand, are brought into proximity. Usually this is only to be found on the second copy that we have of every DNA segment. But if you want to trick the cell, you can offer a piece of DNA that has exactly the same sequence as the ends to the right and left of the point at which it was cut. Now to insert a certain piece of DNA at this site into the genome, you flank it with these sequences. The cell now recognizes the homologous sequence segments and takes this piece of DNA as a template to repair the damage. It thereby incorporates the sequence sections between the homologous areas. In this way, for example, fluorescent proteins or “healthy copies” of genes can be introduced into the genome.

This possibility of making changes to the human genome in a completely new way led to several groups of scientists leading in biotechnology and genetics again calling for a research moratorium in the spring of 2015. Just as in the light of the fundamentally new recombinant DNA in the 1970s, scientists in the field in question are taking this unusual step of trying to impose limits on themselves. This time it was about the possibility of using CRISPR to change the human germ line in such a way that all subsequent generations would carry this change within them. An article in Nature had the unequivocal appeal as the title: “Don’t edit the human germline“.

Only a few weeks after this appeal was published, research by a group of Chinese scientists had actually demonstrated the reasonably successful genetic modification of a human embryo was published. Soon after, a second article has been published that demonstrates the possibility of manipulating human embryos so that they are immune to HIV. And even though in both works, human embryos which cannot develop into viable fetuses due to a genetic trick, were used, many people both inside and outside the research community have the feeling of experiencing a dam break. A broad discourse on the implications of this development seems more necessary today than ever.

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