In addition to small insertions or deletions (indels), the use of the CRISPR/Cas gene scissors can also lead to major structural changes in the DNA. These unintended changes can occur both in the target region (on-target effect) and in other DNA regions, for example, if these are very similar to the target region (off-target effect). Such unintended, structural changes have been found, for example, in experiments on zebrafish in both target and off-target regions (Höijer et al., 2022). Leibowitz et al. (2021) also found extreme unintended changes at the target region when using gene scissors in human cell lines.
Findings of the Geng et al. study
The study by Geng et al. (2022) investigated whether the use of CRISPR/Cas9 with a dual guide RNA can lead to changes at the target region (on-target region). In contrast to simple gRNAs, dual gRNAs cause the Cas9 gene scissors to remove longer DNA sequences from the genome. In this process, a double-strand break is induced at the beginning and end of the target region, thus excising the target sequence. In in vitro experiments on human cell lines (HAP1 and HepG2), the target region studied included two closely spaced genes encoding two tRNAs. tRNAs are essential components in protein biosynthesis. During translation, they act as adapter molecules matching the respective mRNA sequence to mediate the correct amino acids for protein assembly at ribosomes. At the time when the tRNA genes are read by the enzyme RNA polymerase III, this gene segment is in the active state. This loosened state, also known as euchromatic, is made possible by the involvement of so-called histone proteins, which ensure that the DNA is accessible to RNA polymerase III.
After the gene scissors have removed the two selected tRNA genes, neither the RNA polymerase nor the histones should be able to bind in the target region. However, the scientists found in several cell clones which the gene scissors had successfully cut that both elements were nevertheless bound. They then re-examined the changes in detail and also checked which biological processes were still taking place in the altered target region. For these investigations, some methods first had to be newly developed.
Their results showed several changes in the target region in different clones and in both cell lines. Although CRISPR/Cas9 cut in the target region as intended, the scientists were able to show that the excised DNA fragments were not removed from the nucleus. Instead, the cut fragments were reincorporated at the target region both multiple times and, in some cases, in reverse orientation. In addition, they were able to detect the integration of additional exogenous and endogenous DNA fragments as well as a large deletion that occurred near the target region. Furthermore, chromothripsis, an unintentional rearrangement of chromosomes that resulted in remodelling <of the target region, took place in one clone. In terms of protein biosynthesis, they were able to demonstrate that the fragments created from the target region continued to be read and tRNAs were synthesized, albeit at lower levels than before. Moreover, the additionally inserted exogenous DNA fragments were also read and not silenced by the cell, as expected, in which they were more densely packed (heterochromatin).
The results of the study highlight the complexity of human cell repair mechanisms following the use of CRISPR/Cas9 and that different, unintended changes can occur at the target region, with consequences for protein biosynthesis. These results highlight the need to closely examine the target regions for possible changes after using the gene scissors. To do this, the scientists recommend somewhat more extensive methods, such as long-read sequencing (which examines longer stretches of DNA than the usual Sanger sequencing), because the unwanted DNA changes can be very complex, can occur simultaneously, and can be missed by common detection methods such as Sanger sequencing and subsequent PCR verification methods. They also believe that subsequent investigation of the potential biological effects that result from such alterations is essential.
Significance of these findings
With respect to novel genetic engineering, the study demonstrates, firstly, that the use of CRISPR/Cas9 can lead to massive structural changes in the target region, and consequently to unintended effects. Secondly, it shows how crucial the choice and scope of methods are for detecting changes in the target region. The study did not examine non-target regions (off-target regions) because the uniqueness of the target sequence was assumed. Nevertheless, it would be useful to include off-target regions in the future.
Höijer, I., Emmanouilidou, A., Östlund, R. et al. CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations. Nat Commun 13, 627 (2022). https://doi.org/10.1038/s41467-022-28244-5.
Leibowitz ML, Papathanasiou S, Doerfler PA, Blaine LJ, Sun L, Yao Y, Zhang C-Z, Weiss MJ, Pellman D (2021) Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nature Genetics. doi:10.1038/s41588-021-00838-7.