CRISPR/Cas: Methods of genetic engineering involve direct intervention in the genome



The Topic

New genetic engineering techniques (genome-editing) promise to introduce changes in DNA that are supposedly more precise than can be achieved with earlier established techniques of genetic engineering. Until now, methods of genetic engineering have mostly relied upon so-called “shotgun” methods to introduce DNA from other species into the genome. The new gene-editing techniques supposedly make more targeted changes in DNA (genes) possible.

The most important new genetic engineering technology tools are so-called “gene-scissors“ (or “nucleases“), most importantly CRISPR/Cas9. The “gene scissors” target a specific region of the DNA inside the cell with the help of so-called “guide RNAs“; these act as molecular scissors that cut into the DNA. Genetic information can be changed, deleted or new genes inserted at the breakpoint. The nuclease does not necessarily need to cut the DNA in order to function. The structure of the DNA might also be subjected to biochemical changes leading to altered gene expression (epigenetic), or a change in the order of single base pairs (smallest functional unit of the DNA). This is called base-editing.

As a result, genes can be deleted, and/ or silenced, additional genes introduced and /or gene expression altered through epigenetic changes.

The delivery of the nuclease into the cell is achieved via different methods. Normally, as a first step, additional DNA is introduced into cells to produce the molecular scissors CRISPR/Cas. In a second step, the gene-scissors actively change a specific target region of the DNA. Subsequently, in a third step, further breeding is necessary to remove the DNA encoding from the nuclease.

It is also possible to pre-synthesize the CRISPR/Cas protein complex outside the cells and deliver it in its entirety but without additional DNA into the cells. This is less efficient as the enzyme is degraded very rapidly inside the cell.


What is the problem?

Scientific publications indicate that new genetic engineering techniques are less reliable and precise than often claimed. The techniques carry specific risks:

  • The molecular scissors CRISPR/Cas are not completely error-free and can cut at unintended DNA regions that are similar to the target sequence. These effects are known as off-target-effects.

  • Additional DNA might be inserted at the target sequence - as has already been reported in many cases. In particular, the DNA responsible for the synthesis of the nuclease is often inserted.

  • Even if the changes in DNA are successful and precise, the effects of these changes can be very different to what was intended. The composition of the plants’ ingredients might be changed or the risk of infections might be increased due to interaction with other genes. These effects might also be difficult to discover since it is not sufficient to simply analyse the structure of the DNA. On the contrary, it is very often the case that complex biological processes in the cells need to be investigated.

Whatever the case, the new genetic engineering techniques can be distinguished from conventional breeding in their depth of intervention: Conventional breeding techniques always have an effect on the cell or the organisms as a whole, but do not directly intervene in the DNA in the cell nucleus.

In short, gene-editing techniques directly intervene in the cell nucleus, bypassing the 'rules' of gene regulation and inheritance. In contrast, mutagenesis as used in conventional breeding can speed up natural biological mechanisms developed through evolutionary processes but it does not break 'the rules'.

Not only are the processes completely different to random changes in the genome, the effects of the nucleases are fundamentally different, even if no additional DNA is introduced:

  • For example, spontaneous mutations in the genome can be repaired by the cells and restored to their initial state. This is quite different with CRISPR/Cas: If the cell repair mechanism restores the original status then the nuclease will again recognise the target region and be reactivated – this will continue until the original structure of the DNA is destroyed.

  • The new genetic engineering techniques enable multiple changes in the genome, which were not possible with conventional mutagenesis breeding. CRISPR/Cas is able to simultaneously introduce multiple changes that can silence whole families of genes that have developed in an evolutionary process through duplication; the individual genes in the gene families are very similar in their sequences and can act as 'back up' for important information. The gene scissors, however, change
    all genetic regions with the respective target sequence.

  • There are also differences in specific regions in the genome that are not so easily changed. For example, there are special evolutionary conserved regions in the genome where no natural random mutation takes place and there is very little evolutionary change. This applies to genes that are essential for the survival of the organism or the species. It is now possible to change these especially conserved regions by using CRISPR/Cas.


Further information

The regulation of the new genetic engineering techniques (genome-editing) in the European Union is currently being widely debated. Industry wants to exempt many of these techniques from regulation so that they can market their patented seeds and farm animals with no restrictions and increase their profits. For this reason, the new methods of genetic engineering are frequently called “new breeding techniques” or “mutagenesis“. If the new techniques are not classified as genetic engineering, there would be no need for the new organisms to be risk assessed or for them to undergo an approval process. The reasoning behind this is that no foreign DNA is introduced into the organism and that, therefore, it might be difficult to distinguish a gene-edited plant from naturally or conventionally bred plants.

It is indeed more difficult to detect changes in the DNA of gene-edited plants compared to plants that have been genetically engineered by using earlier established techniques. Until now, methods of genetic engineering have mostly been used to insert genes from other organisms into the target organisms and are as such easily detected. These organisms are then called transgenic plants. Genome-edited plants may, but do not necessarily contain DNA from other organisms, which makes it more difficult to detect the genetic intervention. Thus, it is essential that there is an approval process in place that makes methods of traceability mandatory.

Among experts, new gene-editing techniques are often compared to text-editing using a word processing program: New genetic engineering techniques allow the deletion or changes in parts of the natural genome in a similar way to which words or parts of a text in a text editing program can be deleted or changed. Excluding the new techniques from regulation on the basis of this argument is highly questionable. To return to the previous analogy: No one can claim that “editing” the following
sentence makes no difference:

DNA does not describe an organism completely.

Can be changed into

DNA describes an organism completely.

There is a drastic change in the meaning of the sentence even though no other “foreign” or extra words were introduced.

If the new techniques used in gene-editing are not regulated under genetic engineering regulations, the resulting plants and animals will be allowed on to the market without having to undergo an approval process. This means that there will be no way for independent experts to investigate the risks, or any possibility of retrieving the organisms from the environment. There will be no methods for tracing or preventing the organisms from spreading uncontrolled into the environment. The consequences
could impact all future generations.


Further Information:

Table comparison: Differences between Genome editing and mutagenesis