CRISPR Zebrafish

Unintended changes can be very specific

In addition to the intended changes at the target site, the processes of New GE can also trigger unintended genetic changes which differ greatly from those that can be expected from natural processes or conventional breeding methods.

The site of the genetic changes (mutations) and the patterns of genetic change (i. e. the resulting genotypes) can be very different to those which might otherwise be anticipated. This has been shown in zebrafish research.

Prior to publication of the research, it was already known that small changes, such as point mutations or short insertions and deletions, can occur in off-target and on-target regions. Studies in mice and human cell lines also found larger structural changes in on-target regions where, amongst others, large regions of the DNA sequence were deleted or newly inserted. However, it has thus far been unclear whether such large structural changes, such as those described for on-target regions, could also occur at off-target regions. This was investigated in more detail in a zebrafish study.

Various parts of the zebrafish genome were modified using the CRISPR/Cas genetic scissors. The researchers used a version of CRISPR/Cas that increased the possibility of the genetic scissors cutting at off-target sites. Unintended changes were subsequently found, including small changes, such as point mutations and larger changes in the DNA sequence. 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.

According to the study, the unintended genetic changes were inherited in the next generation as well. Surprisingly, in some of the fish not all body tissues were affected to same extent. In addition, deviations from the Mendelian law of inheritance were observed: some gene defects were found to be transferred homozygously, others in a heterozygous manner, without obvious reasons.

Scientists use zebrafish as a model organism 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.

For example, 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 may have a novel and specific risk potential. The differences between naturally occurring processes (or conventional breeding) and NGTs may be easily overlooked but can, nevertheless, have serious consequences. If unintended genetic changes are not detected, they can quickly can spread throughout larger populations.



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