Gene editing can 'refer to which to hit what', four off-target detection methods.

Genome editing technology is the frontier of life science research.
is the most convenient, efficient and widely used in a wide range of genome editing methods with CRISPR/Cas9 systems.
, the off-target effect of CRISPR technology is still the main limiting factor affecting its wide application, how to correctly evaluate and detect off-target effect, and put forward the corresponding strategies to reduce off-target effect, is an important research direction in the field of gene editing research.
recently, Huazhong Agricultural University cotton genetic improvement team a related review, the system summarized the current gene editing tools, gene editing produced off-target effect and type, off-target effect mechanism, animal and plant off-target effect of the problems;
the paper was published online in the international comprehensive journal Advanced Science.
gene editing is good, off-target is a problem compared to the previous research on gene function tools, CRISPR/Cas9 as the representative of gene editing efficiency and accuracy are higher, but also bring sesame a potentially "fatal" problem - off-target effect.
"Gene editing can 'refer to which to hit'."
generally only 20 nucleotide sequences can be located on a gene and targeted knock-out.
at the same time, there are many base fragments in genome sequences that are similar to a specific 20 nucleotide sequences.
gene editing process, fragments that are very close to the target of the guided RNA target are also combined and knocked out, which is called 'off target'.
" Professor of Plant Science and Technology at Huazhong Agricultural University and co-author of the paper, Jin Shuangqi, explained in an interview with China Science Daily.
Jin shuangqi stressed that, because the potential off-target site gene fragments can be combined shorter, so in theory the probability of off-target is relatively high, off-target site is also more.
if the phenomenon of off-target ingestion occurs widely, it will be a serious challenge for plants and animals, the consequences are more serious.
Zhang Yong, a professor at the School of Life Sciences and Technology at the University of Electronic Science and Technology, believes that genome editing technology is important in animal and plant breeding and basic research.
, but the off-target effect and nonspecific shear have an important effect on the reliability and usefulness of the application results and research results.
", there are more reviews of the application and development of gene editing technology, but the comprehensive review of off-target effect is less.
", "For a long time, the academic community has not paid much attention to the off-target effect of plant gene editing and gene editing tools", said Wei Pengcheng, director of the Crop Gene Editing Center of Anhui Province and a researcher at the Rice Research Institute of Anhui Academy of Agricultural Sciences.
four off-target detection methods According to this paper, the gene editing field can now use the following strategies to reduce off-target effect: develop and make rational use of effective tools to predict off-target effect, develop new gene editing system, use high-quality reference genome design target and good gene editing tool delivery system.
" the use of several of these technical strategies, can effectively reduce or avoid off-target.
", said Kim. One of the simplest and most effective methods for
off-target detection is genome-wide sequencing.
the software to predict potential off-target sites and then compare the genomes.
if there is no change in the potential off-target site compared to the receptor material, there is no off-target, there is a change and there may be off-target, which can be further verified by the classical Sanger sequencing method.
" this is a very simple, efficient and comprehensive approach at the moment.
," Kim said.
developing a new gene editing system to avoid off-targeting from the point of view of the technical source, there are some new CRISPR/Cas systems, which have a higher accuracy than the most commonly used CRISPR/Cas 9 systems, which can significantly reduce off-target effects.
Because the genome in gene editing is stitched together through thousands of small fragments obtained through genome sequencing, limited by early sequencing techniques and stitching strategies, many species have errors in earlier versions of the genome, and if such genomes are edited, it is like interpreting a book that is wrong, and it is natural to be misunderstood.
so, without a good reference genome, it is easy to get off target.
" the optimal state is to do genome editing for each species with the reference genome of that species to do the target design.
" Kim said that even in the same species, there are considerable genetic differences between different species and strains, which will have a great impact on off-target, gene editing accuracy.
good gene editing tools should try to avoid or reduce the somatic cell asexuality variation produced by tissue culture, and it is also an important strategy to reduce off-target or improve the accuracy of gene editing. Zhang Yong,
the future prospects of gene editing tools, says the review is very clear about the basic concepts and a comprehensive overview of the methodology.
is also very close to the actual work of research, "not from theory to theory, after reading the work of others to do the literature review, but combined with the first line of work."
" By summarizing the existing research work, the review combs the history of the concept of off-target effect, and makes a more systematic and comprehensive classification of the concept of off-target effect.
in the method level more comprehensive system to explain how to detect the efficiency and feasibility of off-target effect from the experimental point of view, using different gene editing technology, using bioinformatics, AI data analysis and other predictions to evaluate off-target effect, how to reduce off-target effect to improve the accuracy of gene editing and so on.
," Zhang said.
currently, the three most mainstream tools for gene editing are zinc finger nuclease (ZFN) technology, transcription activation act factor nuclease (TALEN) technology and series interval short-form repetition sequence (CRISPR/Cas) system, of which CRISPR/Cas is the most efficient and most used, but its off-target rate is relatively high.
teleN performs best in terms of accuracy.
1990s, the biological community has been using ZFN technology.
taleN technology emerged after 2000.
"early gene editing techniques were protein layers, which meant that the process of releasing proteins was very large in the process of delivering proteins into cells."
" Wei Pengcheng said that although TALEN technology is currently more fidelity and precision than CRISPR/Cas, CRISPR/Cas should be a focus of gene editing technology in terms of energy consumed and the cost of building drugs.
Also believes that although the application of the three mainstream tools is currently in a complementary state, some scientific research institutions will use these three tools at the same time.
the future or CRISPR/Cas has more potential.
"the lack of fidelity and precision currently faced by CRISPR/Cas gene editing tools" should be overcome in subsequent development.
" Wei Pengcheng judged the reason that the early CRISPR protein mainly from natural organisms to screen, and did not make artificial selection as needed, so that can withstand a part of the off-target effect.
once artificial choices are added, fidelity can easily evolve.
", "the future trend will be crispR/Cas technology, will be its existing problems, such as fidelity shortcomings to make up for it."
" (Han Tianqi) Source: China Science Daily, Science.com