Three scientific articles have been published in which the authors report on large-scale fallouts of chromosome genetic material and DNA rearrangements. The scientists' findings, the article says, reinforce concerns about the safety of genome editing technologies.
As a result of a series of experiments in which the modification of human embryos was carried out using the gene editing tool CRISPR-Cas9, scientists have established that this technology can make significant and undesirable changes in the genome at or near the target site of the genome.
The results of the research were published this month on the bioRxiv preprint server, but have not yet been peer reviewed. However, the published articles provide a good indication that some scientists believe there is an underestimated risk in genome editing with CRISPR-Cas9. Previous experiments have shown that this editing tool is capable of causing side gene mutations at a considerable distance from the target DNA site; however, recent studies have revealed changes that occur in areas adjacent to this site and may not be noticed by standard methods.
“For us, precise impacts are more important. They are much more difficult to eliminate afterwards,”says Gaétan Burgio, a geneticist at the Australian National University in Canberra.
Security concerns are likely to be a major topic of ongoing debate over whether scientists can use human embryo editing technologies to prevent genetic diseases. The attitude to this technology raises many questions, since it creates irreversible changes in the genome, which will then be passed on to descendants from generation to generation. "If the technology of editing human embryos for reproductive purposes or editing the so-called germ line can be compared with the first manned flight into space, then the new data obtained by scientists can be likened to a rocket explosion on the launch pad right before takeoff," says Fyodor Urnov from University of California, Berkeley,who is studying genome editing (Fedor Urnov did not participate in any of the above studies).
Unwanted effects
Scientists conducted the first experiments using CRISPR to edit human embryos back in 2015. Since then, several scientific groups around the world have begun to study this technology for precise gene editing. However, such research is still rare and usually highly regulated.
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According to Mary Herbert, a reproductive biologist at Newcastle University, UK, a recent study highlights the following: Scientists know little about how human embryos repair DNA cut with genome editing tools (this is a key step in editing with CRISPR-Cas9). “We need to thoroughly understand what's going on there before we start using DNA-cutting enzymes in it,” adds Mary Herbert.
The first of the preprints was published online on June 5 by developmental biologist Kathy Niakan of the Francis Crick Institute in London and her colleagues. In their study, the researchers used CRISPR-Cas9 to create mutations in the POU5F1 gene, which has a large impact on embryonic development. Of the 18 edited embryos, about 22% contained unwanted changes affecting large DNA regions adjacent to the POU5F1 gene. In particular, there was observed a rearrangement of DNA regions and large deletions of several thousand DNA nucleotides - and this is much more than is usually considered among scientists using this approach.
Another group of researchers, led by stem cell biologist Dieter Egli of Columbia University in New York City, studied embryos produced by sperm that cause a mutation in the EYS gene (this mutation leads to blindness). Scientists tried to correct this mutation using CRISPR-Cas9, but about half of all tested embryos lost large segments of their chromosome (and in some cases the entire chromosome) on which the EYS gene is located.
Finally, a third group of scientists, led by reproductive biologist Shoukhrat Mitalipov of the Oregon Health and Science University in Portland, studied embryos produced using sperm that carry a mutation that causes heart disease. Scientists on this team also seem to have become convinced that editing the genome affects large regions of the chromosome containing the altered gene.
In all of their research, the scientists used embryos only for scientific purposes, and not to induce pregnancy. The leading authors of these three scientific studies, the results of which are reflected in the preprints, declined to discuss their work in detail with the Newsroom of the journal Nature until their articles were published in peer-reviewed journals.
Unpredictable reparation
All recorded changes result from DNA repair, which is carried out using genome editing tools. CRISPR-Cas9 uses a small strand of RNA to direct the Cas9 enzyme to a site with a similar sequence. The enzyme then cuts both DNA strands at this site, and the cell repair systems close this gap.
Editing occurs just during repair: most often, the cell tightly closes this gap with a mechanism that is able to insert or remove a small amount of DNA nucleotides; however, this mechanism works with errors. If scientists insert a DNA template, the cell can sometimes use that sequence to repair the break, resulting in a correct rewriting. However, the cut DNA can also shuffle or lose large chunks of the chromosome.
Previous work using CRISPR technology in mouse embryos and other types of human cells has shown that chromosome editing can cause significant undesirable effects. But, according to Urnov, it was important for scientists to demonstrate their approaches in human embryos, since different types of cells can respond differently to editing the genome.
Such a rearrangement of DNA regions may have been overlooked in many experiments, in which scientists usually try to find examples of unwanted editing, say, a change in one DNA nucleotide, or small insertions or deletions of small fragments of nucleotides. However, in recent experiments, it was specifically large deletions and chromosomal rearrangements near the target site that were specifically investigated. “And the scientific community will take the results obtained even more seriously than before,” says Urnov. "These results are not accidental at all."
Genetic changes
The research teams that have conducted the three studies described above have differently explained the mechanism underlying the rearrangements within DNA. For example, the Egli and Nyakan research teams believe that most of the changes observed in embryos are due to large deletions and rearrangements of DNA regions. However, Mitalipov's group stated that up to 40% of the detected changes were caused by the so-called gene conversion, in which, as a result of DNA repair processes, a sequence is copied from one chromosome in a pair to repair another.
Mitalipov and his colleagues reported similar results back in 2017, but some scientists were skeptical about the fact that gene conversion is common in embryos. The scientists noticed the following: firstly, during gene conversion, the maternal and paternal chromosomes are not located next to each other, and secondly, the analyzes used by the research team to determine the gene conversion could reveal other chromosomal changes, including deletions.
Egli and his colleagues in their preprint wanted to directly experimentally verify the presence of gene conversion, but could not detect it. Burjo notes that the experiments described in Mitalipov's preprint are similar to those conducted by his research team in 2017. According to Jin-Soo Kim, a geneticist at Seoul National University and co-author of the Mitalipov preprint, DNA breaks in different parts of the chromosome can be corrected in some other way - this, in his opinion, is one of the possible solutions to the issue …