Nature (UK): editing of the gene of the human embryo using a technique CRISPR leads to large-scale disorder in the chromosomes

In a series of experiments in which modification of human embryos was performed using the gene editing tool CRISPR-Cas9, the researchers found that this technology can make a significant and unwanted changes in the genome at a site of the target genome or close to it.

The results of these studies were published in the current month on the bioRxiv Preprint server, but have not yet passed peer review. However, published articles give a good idea of what, according to some scientists, there is an underestimated risk arising from the genome editing using CRISPR-Cas9. As shown by earlier experiments, this editing tool can cause adverse genetic mutations at a considerable distance from the site of the target DNA, but recent studies revealed changes in the surrounding land areas and can be seen by standard methods.

“For us, it’s more accurate effects. Subsequently fix them a lot harder,” says Gaetan of Burgo (Gaétan Burgio), a geneticist from the Australian national University in Canberra.

Probably, concerns about security will be the main theme of the ongoing debate about whether scientists use the technology of editing of human embryos to prevent genetic diseases. The attitude to this technology raises many questions because it helps to create irreversible changes in the genome, which will then be transmitted to descendants from generation to generation. “If the technology of editing of human embryos for reproductive purposes or edit so-called germ line can be compared with the first manned flight into space, the new data scientists can be likened to the explosion of the rocket on the launch pad just before takeoff,” says Urnov, Fyodor (Fyodor Urnov) from the University of California at Berkeley, who has been studying the editing of the genome (Fyodor Urnov did not participate in any of the above studies).

Adverse effects

The first experiments using CRISPR to edit the human embryos, scientists have spent more in 2015. Since then, several research groups in different countries of the world have begun to explore this technology, designed for precise editing of genes. However, such studies are still scarce and usually strictly regulated.

According to specialist in reproductive biology Mary Herbert (Mary Herbert) from Newcastle University (UK) in recent research work emphasizes the following: scientists, little is known about how human embryos occurs mending DNA that has been cut with the tools for genome editing (this is a key step in editing with CRISPR-Cas9). “We need to thoroughly investigate what is happening there, before we start to use it enzymes, cutting DNA,” adds Mary Herbert.

The first paper was published online June 5, a specialist in the field of developmental biology Katie Niacin (Kathy Niakan) from the Institute of Francis Crick, London, and her colleagues. In their study, the scientists used CRISPR-Cas9 to generate mutations in the gene POU5F1, which has a great influence on embryonic development. Of the 18 embryos edited about 22% contained unwanted changes, affecting large stretches of DNA adjacent to the gene POU5F1. In particular, there was observed a restructuring of the DNA regions and large deletions of several thousand nucleotides of DNA and it is much more than usually assumed among scholars using this approach.

Another group of researchers, led by expert stem cell biologist Dieter egli (Egli Dieter) from Columbia University in the city of new York, studied the embryos, protezirovanie sperm, which cause mutation in the gene EYS (this mutation leads to blindness). Scientists tried to correct the mutation using CRISPR-Cas9, however, about half of all tested embryos lost large segments of their chromosomes (and in some cases the entire chromosome), which is the gene EYS.

And finally, the third group of scientists, led by a specialist in reproductive biology Shukhrat by Mitalipov (Shoukhrat Mitalipov) from the University of Oregon health Sciences in Portland, studied the embryos obtained using sperm-carriers of the mutation, which causes heart disease. Scientists from this team, too, apparently, was convinced that the editing of the genome affects a large region of the chromosome containing the gene.

In all their research, the scientists used embryos only for research purposes, not to induce pregnancy. Lead authors of these three scientific research, the results of which are reflected in the preprints, refused to discuss his work with the news Department of the journal “Nature” as long as their articles are not published in peer-reviewed journals.

Unpredictable repair

All the recorded changes arise as a result of DNA repair, which is carried out using tools for genome editing. CRISPR-Cas9 with the help of small chain RNA directs the Cas9 enzyme to the site (site — a term applied to any small parts of the genome — approx. transl.) with the same sequence. Then an enzyme cuts in both strands of DNA and mismatch repair system cells eliminate this gap.

The edit occurs just in time for reparations: the most common cell tightly seals the gap through a mechanism that is able to insert or delete a small number of nucleotides of DNA; however, this mechanism is not working correctly. If scientists insert DNA template, the cell can sometimes use this sequence to fix the gap that leads to a valid rewriting. However, the cut DNA can also shuffle or lose large parts of chromosomes.

In previous work, where the CRISPR technology has been used on embryos of mice and in other types of human cells, it has been shown that editing of chromosomes can cause significant unwanted effects. But, according to Urnov, the scientists it was important to demonstrate their approaches on human embryos, since different cell types can respond differently to edit the genome.

This alteration of DNA could be seen in many experiments in which scientists usually try to find examples of undesirable editing, say, a change in one DNA nucleotide or small insertion or deletion of small fragments of nucleotides. However, in recent experiments were studied of large deletions and chromosome rearrangements in the vicinity of the target. “And the scientific community would react to the obtained results even more seriously than before, says Urnov. — These results are not coincidental.”

Genetic changes

A team of scientists who conducted three studies described above, different explained the mechanism underlying the rearrangements in DNA. For example, research teams egli and Nyakana believe that most of the changes observed in embryos, due to large deletions and rearrangement of DNA. However, the group Mitalipov said that up to 40% of the detected changes were caused by so-called gene conversion, in which the result of the repair processes of DNA copy sequences from one chromosome in a pair to repair the other.

Mitalipov and his colleagues reported similar results in 2017, but some scientists were skeptical of the fact that gene conversion is common in embryos. Scientists have noticed the following: first, during gene conversion of maternal and paternal chromosomes are next to each other, and secondly, the tests used by the team of researchers to determine gene conversions, could reveal other chromosomal changes, including deletions.

Egli, along with colleagues in his paper wanted to experimentally conrm the presence of gene conversion, but are unable to detect it. Burgo notes that the experiments described in the paper Mitalipova similar to those carried out by his research team in 2017. According to Jin-su Kim (Jin-Soo Kim), genetics of Seoul national University and co-author of a Preprint Mitalipova, DNA strand breaks in different locations on the chromosome can be corrected and somehow differently — that is, in his opinion, one of the possible solutions to the issue.