CrispR-Cas9 chain replacement amplification technology for nucleic acid hypersensitivity testing

Recently, Zhou Wenhua, Ph.D., Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, and others made new progress in the field of temperature-amplification gene detection, such as CRISPR.
related work", "A CRISPR-Cas9-triggered strand edified amplification for ultrasensitive DNA detection" (A CRISPR-Cas9 chain replacement amplification technology for nucleic acid hypersensitivity testing) is published in the international journal Nature-Communications (Nat. Commun. 2018, 9, 5012)。
the paper co-first author is Zhou Wenhua and research assistant Hu Li, and the communication author is researcher Yu Xuefeng.
nucleic acid molecular detection has been widely used in precision medicine, food safety and quarantine, public safety monitoring and other fields.
Although PCR-based nucleic acid detection technology is widely used in specialized testing facilities, it is not suitable for basic detection and home detection due to its complex operation, expensive instruments, and multiple temperature change processes.
in order to overcome the shortcomings of PCR technology, a kind of detection technology that does not rely on variable temperature, that is, i.e. temperature amplification detection technology has been widely concerned.
however, the current isometric amplification technology in terms of sensitivity, specificity and anti-jamming degree still have their own inherent defects, and mature iso-iso-iso-amplification technology core patents are mostly in the hands of foreign companies.
therefore, there is an urgent need to develop a new temperature amplification testing technology with better performance and fully independent intellectual property rights to meet the growing demand in the field of nucleic acid testing in China.
CRISPR/Cas9 as a gene-targeted editing technique derived from the acquired immune system of pronuclear organisms, has attracted a lot of scientific attention and investment interest since its discovery.
however, due to the complexity of human body and ethical disputes, clinical gene therapy based on CRISPR technology still faces many difficulties and challenges.
on the other hand, because of the advantages of simple operation, high sensitivity and strong anti-jamming, the technology has begun to be applied in the field of in vitro nucleic acid molecule detection.
at present, the nucleic acid detection method based on CRISPR technology still relies on traditional PCR technology or iso-temperature amplification technology to amplify the target molecules, and its innovation and application range still need to be improved.
in view of these current situations, Yu Xuefeng's team innovatively proposed the use of CRISPR system effect protein Cas9 in the process of binding with the target nucleic acid molecules unique conformational changes, as a chain to replace the switch of iso-temperature amplification reaction, efficient start the index double-amplification for target nucleic acid molecules (CRISDA technology).
crissdDA technology has many unique advantages over traditional PCR and other isometric amplification technologies.
first, the technology is highly sensitive.
based on CRISPR technology for good anti-jamming, the technology can be used to efficiently amplification detection of target nucleic acid molecules at aM (10-18M) concentrations under complex background conditions.
second, CRISDA technology is highly specific.
through the special optimization of mechanism, the technology is a good way to avoid the widespread off-target ingestive effect in the traditional CRISPR gene editing technology, and can achieve the detection of single nucleotide polymorphism (SNP) for very low concentrations of target nucleic acid molecules. Third,
, CRISDA technology is extremely universal.
in the detection reaction for different target sites, the required amplification primer design is simple, no optimization, can quickly realize the development of the new site detection reaction system. In addition to
, the technology detection process is completely equal temperature, and from room temperature to 42oC range are maintained in a good amplification detection effect, can fully meet the actual detection of new targets in the need.
, the team has used this technology to successfully detect breast cancer-related single nucleotide site mutations in the human genome, and in wild soybeans successfully detected three-tenths of the genetically modified soybeans.
Figure 2: Specific amplification testing of target molecules of aM magnitude is carried out using CRISDA technology.
(a) amplification system design and (b) specific amplification testing of synthetic DNA fragments using CRISDA technology.
(c) specific amplification testing of DNA fragments from the human genome using CRISDA technology.
(d) specific amplification testing of specific regions of the human genome using CRISDA technology.
Figure 3: SNP amplification testing of target molecules of aM magnitude is carried out using CRISDA technology.
(a) sequencing verification of rs3803662 sites in the genomes of different cell lines; at the same time,
, because of the unique advantages of CRISDA technology and beyond the traditional PCR technology application prospects, with this technology as the background of the "CRISPR-Cas system-based nucleic acid high-sensitivity rapid temperature detection technology" project in the Chinese Academy of Sciences in the first "first cup" future technological innovation competition, from hundreds of projects stand out, won the final award, and has attracted a number of investment institutions to negotiate industrialization.
currently, the team is applying the technology to detection of sudden or new infectious diseases, such as new influenza, African swine fever, and is actively developing relevant testing kits.
the research was supported by the National Natural Science Foundation of China, the Shenzhen Science and Technology Program International Cooperation Research, the Chinese Academy of Sciences Frontier Science Research Focus Program, the Shenzhen Science and Technology Research Program, the Lihua Hume Trust in the United Kingdom and the Hong Kong Research Grant Project.
Source: Shenzhen Advanced Technology Research Institute.