How to detect gene editing off target? The risk of gene editing off target is almost inevitable.

Perhaps you are suffering from a single genetic disease, such as haemophilia, alcoagulation, and so on.
one day, the doctor will tell you that you can use gene editing technology to "cut off" the "bad gene" that makes you suffer? Don't worry, gene editing this is called "God's scalpel" magic technology, actually also miss the time, then how should we face it... Recently, a technique called GOTI has received widespread attention, published in the journal Science, and developed by the Institute of Neuroscience of the Chinese Academy of Sciences in collaboration with researchers from domestic and foreign research institutions to accurately and sensitively detect whether gene editing methods have an off-target effect (referring to the wrong editing).
, how do scientists detect gene editing off target? Why is it so hard to detect off-target? How will off-target technology develop in the future? Professor Liu Dong of Nantong University, Jiangsu Province, explained in detail to the reporter of Science and Technology Daily.
contrast sequencing detection off-target operation difficult gene editing technology refers to the ability to "edit" the target gene, such as the specific DNA fragments of knock-out, knock in, etc.
and CRISPR/Cas9 technology has been available since its incomparable advantage, making it easier for scientists to "delete" and "copy" and paste on their cells.
as human genetic editing ability becomes stronger, gradually, worry will come, will gene editing fail to create a Hulk-like monster? Such a possibility is not non-existent.
, it is difficult to avoid the "off-target" effect because gene editing tools such as CRISPR are so powerful.
if it is used in experimental animals such as plants or zebrafish and mice, it is easier to accept even the consequences of error, or even abandon it.
But in human embryo editing, even a very small probability of off-targeting can cause a lot of problems because you can't give up an embryo or a living person for another test.
, therefore, the detection of gene editing tools is off target, has become a scientific community must overcome a subject.
", the traditional detection method, in short, is sequencing and analysis.
" Liu Dong said, before that, people have introduced a variety of detection off-target programs.
previous methods were not suitable for detecting individual nucleotide differences in the body.
he wrote with a text edit: "We delete a word when we edit a paragraph of text, how can we be sure that the word was deleted is the same as expected?" Ideally, you need to compare it with an original document.
", in gene editing, scientists divide the sample cells into two parts, one for gene editing, one for not to do, and finally to determine two sets of gene sequences, in addition to the changes in the target gene, other genes should theoretically be the same, if there is a difference may be caused by gene editing off target.
"But life is different, and because of the polymorphism of single nucleotides, we can't find the original sample for comparison, which is also a characteristic of biodiversity."
," Liu dong said.
, a large number of samples are required for gene sequencing, and the sample cells must be in vitro amplification, i.e. large replication.
in the amplification process, there is a probability of DNA mutations, which are called "amplification noises", which make it more difficult to distinguish between off-target and mutations.
, it is theoretically feasible to detect whether gene editing is off target, but it is difficult to operate in practice.
why the new "judge" can find out that although the new generation of gene editing, represented by CRISPR/Cas9, is known for its accuracy, each gene editing operation is essentially thousands of gene editing tool molecules that have made multiple edits to cells, which are unavoidable but low in probability, leading to extremely weak off-target signals that drown out the powerful background noise.
how to remove background noise is what scientists want to do, and the Chinese Academy of Sciences Institute of Neurology's Yang Hui laboratory method is very novel.
this off-target test, called GOTI, is an experiment using mouse embryos.
the researchers edited a fission ball and labeled it with red fluorescent protein as the fertilized eggs of mice split into two cells.
mouse embryos developed to 14.5 days, the entire mouse embryo was digested into a single cell, using flow cell sorting technology based on red fluorescent protein, the selection of gene-edited cells and no gene-edited cells, and then the whole genome sequencing to compare the two sets of differences.
"It's hard to find two mice with exactly the same genes, but the experimental and control groups came from the same fertilized egg, and the genes were theoretically identical, so that by comparing them, they could find out if the gene editing was off target."
," Liu Dong told reporters.
" the subtlety of this technique is that it solves the problem of small samples but high accuracy.
" Liu said, "the small amount of DNA in a single cell is not enough for sequencing analysis."
but the scientists fluorescently labeled the fertilized egg for two cells, then waited for it to develop into an embryo and then "split" it into enough single cells to sequence it, eliminating the need for in vitro amplification to amplification the noise problem.
using this technique, crispR/Cas9 derivative technology BE3 monobase editing produces a large number of off-target mutations, confirming that some gene editing technology represented by BE3 has an unpredictable risk of off-target.
discovery led to a re-examination of the single-base mutation technique that was supposed to be "particularly safe and hardly off target."
how ethical issues can be avoided remains to be seen, but industry experts have doubts about the feasibility of the technology in experimental animals and the ethical implications for humans.
countries have long regulated the use of gene editing in humans with a variety of ethical principles, one of which is that it is necessary to have the approval of a national government ethics body, and can not allow genetically modified babies to be born, even if the use of human embryos for research, is generally limited to 14 days of embryos, that is, embryos development to 14 days after the destruction, can not develop into human.
the test, which requires embryos to mature, is illegal in many countries, let alone waiting for the embryo to develop into adult form.
, experts believe the technology opens a new window into gene editing, but it still needs to be refined.
" precision is hard to do in life science.
because from a microscopic point of view, the continuation of life is based on a dynamic process of weak forces, genetic change is a probability problem, want to be very accurate, I think it is not true in theory.
" Liu Dong thinks.
GOTI found that the single-base editing technology has off-target problems, should not give up the research on this technology? The answer is no.
many people's genetic diseases are caused by monobase mutations.
data show that there are 7,000 rare diseases worldwide, 80 per cent of which are monogeny genetic diseases and 50 per cent in childhood.
", for example, the gene therapy experiments we are doing for deafness, where many of the phonophorescous deafness is caused by a single gene mutation, and experiments in mice have shown that repairing a single-base mutation gene can restore hearing.
," Liu said. Liu Dong, a
, believes that future gene editing for clinical treatment, it is necessary to establish industry standards, and very strict.
, however, just as the drug has side effects, surgery may fail, and the risk of gene editing off target is almost inevitable, as long as the probability is within acceptable limits.
" that is, when hope is greater than risk, someone will do it, even when there is no choice.
" Source: Science Daily.