In recent years, the CRISPR/Cas9 gene editing technology is revolutionizing the entire research community. The technology uses the CRISPR system to inject Cas9 protein and guide RNA into mouse fertilized eggs, enabling gene knockout and injection of Donor DNA. Gene knock-in can be achieved, and even internationally renowned academic journal Genome Biology has pointed out that this new technology will soon replace the genetic modification of embryonic stem cells, and it seems that the end of mouse embryonic stem cell genetic modification technology is inevitable.

However, is this the case?

Off-target effect remains controversial

The CRISPR/Cas9 gene editing tool called "Magic Scissor" is highly anticipated for genetic engineering applications. However, the controversial old problem of off-target effects has been affecting the application of the CRISPR/Cas9 system. It has been reported in the literature that the cleavage of CRISPR/Cas9 is through the pairing of 20 bp base between the gRNA and the genomic position, and the 5' end of the paired base needs to have a PAM structure, which is then guided by the action of Cas9, but the CRISPR/Cas9 and the target position. The specificity of point recognition is mainly dependent on the pairing of gRNA with 10-12 bp base near PAM, while the other recognition of the mismatched target site away from 8-10 bp of base at PAM is not obvious. This study directly illustrates CRISPR /Cas9 has severe off-targetity, which means that the technology can undergo non-specific cleavage, causing mutations in the non-targeting sites of the genome, which will cause uncertainty in the results of the study.

The genome-wide sequencing analysis of the study "Unexpected mutations after CRISPR-Cas9 editing in vivo" published in Nature Methods last year showed that CRISPR gene editing introduces hundreds of unpredictable mutations into the genome, the large-scale off-targets pointed out in the article. The problem raised a sharp challenge to the application of CRISPR, but it reversed on March 30 this year and was retracted. It is worth noting that the first author of this article, Kellie A Schaefer, and the co-first author, Wen-Hsuan Wu, agreed to withdraw the manuscript, while the other four authors, including correspondent Alexander G Bassuk and Vinit B Mahajan, disagreed. Retractions. This is different from the usual retraction. Most of the general retraction seems to be the resignation of the author of the communication. The author of this controversial article insisted on his opinion. Obviously, this article also has huge differences within the researchers.

However, some studies have shown that the problem of off-targeting of CRISPR is not so serious: the study in the journal BioRxiv "Whole genome sequencing of multiple CRISPR-edited mouse lines suggests no excess mutations." pointed out that many small clones using CRISPR/Cas9 technology gene editing The results of whole-genome sequencing of the mouse strains show that the CRISPR/Cas9 technology can accurately edit the genome at the biological level without introducing many unexpected off-target mutations.

From these controversial studies, it can be seen that the off-target effect still needs to be carefully considered when applying CRISPR/Cas9 technology. On the whole, ES target technology with accurate modification, stable effect and very low target rate is still a better choice for researchers.

CRISPR/Cas9 technology is currently difficult to handle complex gene editing

On the other hand, for complex genetic modifications (such as conditional knockouts, conditional point mutations, and large-segment knock-ins, etc.), CRISPR is congenitally deficient because it requires homologous recombination of Donor DNA to a specific location in the genome of the fertilized egg. And the efficiency of homologous recombination is very low. On the contrary, ES target technology has obvious advantages in this respect due to its wide range of flox region, high tolerance to complex structures and high success rate of target.

The dispute over the patent of CRISPR/Cas9 is still controversial, and there are major hidden dangers in commercial applications.

Another issue worthy of attention for the CRISPR/Cas9 technology is its patent use. The CRISPR/Cas9 patent is based on the research results of many organizations, and the property rights are not clear. There has always been a dispute over ownership.

In early 2017, Broad Institute and co-authors were authorized to publish CRISPR patents on genome editing of eukaryotic cells (including human cells), while UC Berkeley and co-authors patented the use of CRISPR technology to cells. In the -free system, it is not a genome editing involving eukaryotic cells. However, the latter is not satisfied with the outcome of this decision. The unsatisfactory result is the initiation of the patent proceeding. Through this procedure an inventor can take over the patent of another inventor.

According to data from consulting firm IPStudies 2017, there are currently 763 Cas9-related patents, some of which claim to have certain patents for editing the CRISPR/Cas9 gene. Over time, the owners of these patents Maybe also try to maintain their rights.

The dispute over the patents of CRISPR has caused great confusion for some commercial organizations. First of all, this means that there is a large legal risk in purchasing technical services and products based on CRISPR/Cas9 technology from commercial organizations that are not licensed by the CRISPR/Cas9 technology. Secondly, it is difficult to guarantee purchases from a patent owner. Patents cover all application needs.

The number and quality of articles published using CRISPR/Cas9 technology is lower than articles published by ES Targeting Technology

Search for knockout "embryonic stem cell" or "ES cell" by Google Scholar 2013-related articles about CRISPR/Cas9 gene knockout and ES targeting technology. Found about 17300 results, search for knockout "crispr" or "cas9" About 16100 results.

According to the two search results, 100 articles in the search list were randomly sampled. After statistical analysis, it was found that the average impact factor of articles published by ES targeting technology was 2.1 IF higher than that published by CRISPR/Cas9 technology. Therefore, regardless of the quality or quantity of the article, the current influence of CRISPR/Cas9 technology is still weaker than the gold standard ES target technology. However, it cannot be denied the potential impact of the future of CRISPR/Cas9 technology.

In view of the above aspects, the CRISPR/Cas9 gene editing technology that has been in the hot spot in recent years has seen many similar "crisis". Although there are many advantages, the academic community still needs to maintain a calm attitude. Researchers from different countries and regions are exploring the field of CRISPR. Perhaps this series of events is not a crisis for CRISPR. It is an important force to promote its development. We also hope that scientists can continue to explore and optimize the process. Genome editing techniques are more widely used in clinical disease treatment.

This article is from the Internet, and the purpose of reprinting is to share insights. If there is any infringement, please let me know!

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