CRISPR Zebrafish

Unintended changes can be very specific

In addition to the intended changes at the target site, CRISPR/Cas applications can also trigger unintended changes in the genome, known as off-target and on-target effects.

Changes at the target site are referred to as on-target effects. These describe both intended and unintended changes to the target sequence. Off-target effects occur at regions of the genome which are similar to the actual target sequence. The genetic scissors can cut these sites - and thus cause unintended changes. In the case of off-target effects, the genetic scissors confuse the actual target site with non-target sites.

All these changes, including unintended changes, have one thing in common: the patterns of genetic change (i.e. the resulting genotypes) may be novel or specific to a particular case. This results from the CRISPR/Cas mechanism of action. Research on zebrafish, amongst others, shows that this is also valid for unintended changes. Consequently, the risks associated with the new genetic engineering methods are different to those known from previous breeding methods.

Until now, it was known that small changes, such as point mutations or short insertions and deletions, can occur in off-target and on-target regions. In studies on mice and human cell lines, larger structural changes were also found in on-target regions. For example, large regions of the DNA sequence were deleted or newly inserted. However, it has thus far been unclear whether such large structural changes, as described for on-target regions, could also occur at off-target regions. This has now been investigated in more detail in a study on zebrafish.

Various parts of the zebrafish genome were modified using the CRISPR/Cas genetic scissors. The guide RNAs (signposts which guide the genetic scissors to target sequences in the genome) used by the scientists were relatively imprecise, thus increasing the possibility of the genetic scissors cutting at off-target sites. Unintended changes including small changes, such as point mutations and larger changes in the DNA sequence, were found there. For example, 903 base pairs (these are DNA letters) were deleted at one off-target region, thus shortening a large part of a gene that was not supposed to be changed at all. Such changes can have effects on a wide variety of metabolic processes and should, therefore, be closely examined.

Scientists use zebrafish as model organisms in basic research to investigate fundamental mechanisms. These fish are not intended to be marketed. However, the findings from such studies can be extended to other target organisms, and can also be relevant in regard to risk assessment.
It has not yet been clarified whether similar, large structural changes also occur at off-target sites in CRISPR/Cas applications in plants. However, it is likely, as similar effects have already been reported in on-target regions in genome-edited rice plants.

This example from basic research shows: new genetic engineering methods are error-prone and can induce a variety of unintended changes. These have a novel and specific risk potential.


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