Major unintended restructuring of the genome at or in the vicinity of the target sequence are known as so-called „on-target effects“. Major restructuring in the genome can be, for example, large deletions, that is, very large pieces of DNA are deleted or large DNA sequences are unintentionally inserted at the target sequence.
In previous studies, such large-scale restructuring of the genome after application of CRISPR/Cas9 had already been described in mice and human cells (e.g. original publications: Adikusuma F, Piltz S, Corbett MA, Turvey M, McColl SR, Helbig KJ, Beard MR, Hughes J, Pomerantz RT, Thomas PQ (2018) Large deletions induced by Cas9 cleavage. Nature 560 (7717): E8-E9. doi: 10.1038 / s41586-018-0380-z and Kosicki M, Tomberg K, Bradley A (2018) Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36 (8): 765-771. doi: 10.1038 / nbt.4192). In this body of work with human cell lines, the DNA encoding the gene scissors was stably integrated into the genome of the cells and the gene scissors formed continuously. The target sequences were altered by the activity of the gene scissors and the cell’s own repair mechanism NHEJ.
By contrast, in this current study by Weisheit et al. (2020), the occurrence of unintended on-target effects after CRISPR/Cas9 tests in human induced pluripotent stem cells (iPSC), in which the gene scissors are only temporarily formed in the cells, was investigated for the first time. The DNA encoding the gene scissors is therefore not stably inserted into the genome, but only temporarily present in the cells. It was also investigated whether such large rearrangements also occur when another cell repair mechanism called HDR is activated. HDR is used in SDN-2 and SDN-3 applications by CRISPR / Cas9 to incorporate DNA templates into the target sequence. In the study, a variant of the APP gene was inserted into the target sequence using HDR. This gene is involved in the development of Alzheimer’s disease.
The study describes a method for detecting on-target effects, which the authors call quantitative genotyping PCR (qgPCR). To date, other methods such as long-range PCR have been used to identify large rearrangements, but these are quite expensive and complex to carry out. With qgPCR, a classic PCR is carried out in which the target sequence is replicated in the genome-edited cells. In addition, a fluorescent probe that can bind to the target sequence is designed. By duplicating the target area in the PCR, the glowing probe can be used to determine whether one or two copies of the target sequence are present in the cell. This means that deletions on the target sequence can be determined very quickly and efficiently.
The scientists found on-target effects in up to 40% of the cells examined, which have incorporated DNA templates into their genomes through activation of the HDR. The scientists also found large deletions in the target sequence in 50% of the iPSC cells in which the target sequence was changed by activating the NHEJ repair. These deletions could remain undetected without a suitable examination method, since standard PCR cannot determine whether two or one copy of the target area are present. If a copy of the gene is accidentally deleted after a CRISPR/Cas application, the results of functional studies can be misleading.
An effect called loss-of-heterozygosity (LOH) is also described: genetic variants naturally occur at certain points in the genome. This means that an individual can carry different variants of a gene (a so-called allele) at individual gene regions, which is known as heterozygosity. The investigation showed that, in addition to the built-in DNA template, these small differences in the genetic material were lost and deleted DNA areas were simply replaced by the second, non-deleted copy of the gene. There are then two identical gene copies where originally two different alleles were present. This can affect large parts or even entire arms of chromosomes. The authors of the study do not have a conclusive explanation for this effect.
Finally, the authors call for suitable quality controls that need to be carried out after genome editing in order to identify unintended on-target effects at an early stage.
Comment: Furthermore, it should not be overlooked in the debate that large rearrangements could also occur in parts of DNA that are not in the direct vicinity of the target sequence, but rather further away from it.