CRISPR nucleic acid detection technology

The CRISPR-Cas system, derived from the adaptive immune mechanism of prokaryotes, quickly stands out from many competitive gene editing technologies due to its high specificity, strong developability, simplicity and efficiency, and is not only an in vivo gene editing technology It has brought revolutionary breakthroughs and opened up new directions in the field of in vitro diagnostics. In recent years, many fast, portable, economical, and efficient in vitro diagnostic technologies based on CRISPR-Cas systems have emerged. These technologies have shown good performance in pathogen detection, and are used in tumor genetic diagnosis, genetic disease screening, and transplant rejection detection. Cancer detection, microbial resistance detection, environmental microbial detection and other fields also have great potential.

The earliest CRISPR nucleic acid detection technology is to use amino acid-modified dcas9 protein to fuse with egfp fluorescent protein, and to specifically bind to the target sequence under the guidance of a specific guide RNA. This method is time-consuming, expensive, complicated to operate, and difficult to apply in practice.

The turning point of CRISPR nucleic acid detection technology came in 2016, when Zhang Feng's team found that cas13a has bypass cleavage activity. When cas13a binds to the crRNA with the target recognition region, it can specifically recognize the target single-stranded RNA and cut the target RNA. At the same time, the alternative cleavage activity of the enzyme is activated to nonspecifically cleave nearby single-stranded RNAs. Using this technology, in 2017, the first relatively simple and practical CRISPR nucleic acid detection technology, sherlock, was born. At the same time, the bypass cleavage activity of cas12a was also found. Different from cas13a, cas12a can specifically recognize and bind double-stranded DNA under the guidance of guide RNA, and obtain the bypass cleavage activity on single-stranded DNA. On the basis of this technology, detectr and holmes detection methods have been published successively.

Introduction to CRISPR-Cas System

 The CRISPR-Cas system is a special immune system of prokaryotes, which exists in about half of bacteria and almost all archaea. The adaptive immune function mediated by CRISPR-Cas can be summarized into 3 processes: Adaptation, maturation of CRISPR RNA (crRNA) and targeted interference [1]. According to the types of Cas effector proteins, CRISPR-Cas systems can generally be divided into 2 categories, including 6 types and 48 subtypes [2]. A class of CRISPR-Cas systems (a class of systems) includes three types: I, III, and IV, represented by Cas3, Cas10, and Csf1, respectively, which utilize CRISPR-associated virus defense complexes composed of multiple Cas proteins. complex for antivirus defense, Cascade) to form an interference mechanism to cleave target nucleic acid sequences. About 90% of CRISPR-Cas systems belong to a class of systems [3]. Type II CRISPR-Cas system (Type II system) also includes 3 types: II, V and VI, which are represented by Cas9, Cas12a (Cpf1) and Cas13a (C2c2), and recently found that Type V also includes a relative molecular mass The smallest Cas14.

Pathogen nucleic acid detection method based on CRISPR-Cas9

Pardee et al. developed a nucleic acid sequence-based amplification (NASBA) CRISPR cleavage (NASBA CRISPR cleavage, NASBACC) assay to detect Zika virus (ZIKV) in blood samples and use it to identify SNPs Ability to distinguish American and African ZIKV strains. The method firstly uses NASBA technology to reverse transcribe RNA and amplify enough dsDNA, Cas9 specifically binds and cuts the target dsDNA, and finally uses the toehold sensor to read the result through the color change on the test paper. Huang et al. paired CRISPR-Cas9 with exponential amplification reaction (EXPAR) to establish the Cas-EXPAR method. Cas9 cleaves ssDNA molecules into short fragments (X fragments) with the help of independent oligonucleotides containing PAM sites, the X fragments are cleaved by the endonuclease NEase and released from the EXPAR template by exogenous DNA polymerase , and finally, the X fragment is cyclically amplified, and the amplified product is detected by real-time PCR (RT-PCR), which can rapidly and highly sensitively detect Listeria DNA. Wang et al. constructed a CRISPR typing PCR technology to identify PCR products by gel electrophoresis or receiving fluorescent signals, which can be used to detect two high-risk types of human papilloma virus (HPV) 16 and 18. Quan et al. established a next-generation sequencing method to discover low-abundance sequences by hybridization, which has been applied to the diagnosis of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus infections and the detection of Plasmodium falciparum resistance genes. The method utilizes sgRNA-Cas9 complex to cleave phosphatase-treated pathogen genomic DNA or complementary DNA, and then ligates the cleaved product to a universal sequencing adapter, and then performs next-generation sequencing on the amplified product. Wang et al. developed a CRISPR-Cas9-mediated lateral flow assay that can detect Listeria or African swine fever virus DNA within 1h.

The dCas9 genetically engineered from Cas9 has DNA-specific binding ability but no cleavage activity. Zhang et al. fused the split two halves of luciferase with dCas9 and designed a pair of sgRNAs targeting the upstream and downstream sequences of DNA. When the two halves of luciferase are adjacent, the split luciferase activity will be restored. This method Known as a PC reporter, it is used to detect Mycobacterium tuberculosis. Guk et al. established a CRISPR-mediated DNA fluorescence in situ hybridization method to detect MRSA, which uses SYBR Green I as a fluorescent probe and dCas9 to specifically capture target DNA. Another type of modified Cas9 protein mutant Cas9n has a nuclease activity domain inactive, so it can only cut ssDNA. [16] used Cas9n to establish a Cas9n-based amplification reaction to detect the DNA of Salmonella typhimurium, Escherichia coli, Mycobacterium smegmatis and other bacteria.

Pathogen nucleic acid detection method based on CRISPR-Cas12/Cas13

Chen et al. established an endonuclease-targeted CRISPR trans reporter detection system (DETECTR) using LbCas12a. Using the Cas12a-crRNA complex to bind and cleave PAM-complementary dsDNA and PAM-independent ssDNA activated collateral cleavage activity, HPV types 16 and 18 can be effectively detected in clinical samples. The one-hour low-cost, multi-purpose and high-efficiency system is similar in principle to DETECTR and has the characteristics of rapidity, economy, multiplexing, and high efficiency. Unlike DETECTR, it is not only used to detect DNA viruses, such as pseudorabies virus; such as Japanese encephalitis virus. Dai et al. established an electrochemical sensor based on CRISPR-Cas12a for the first time. This method uses the reporter method of electrochemical current to detect target nucleic acid, which can detect the DNA of HPV16 and parvovirus B19. Teng et al. developed a Cas12b-mediated DNA detection method for the detection of HPV16 DNA in human plasma. Wang et al. developed a Cas12-mediated lateral flow assay for rapid and accurate detection of African swine fever virus nucleic acid.

Pathogen nucleic acid detection based on Cas13

The organic combination and integration of microfluidic technology and CRISPR-Cas13-based detection methods makes it possible to perform simultaneous detection of hundreds of samples or multiple pathogens. Qin et al. developed a Cas13a-based automated microfluidic POCT device for simultaneous detection of Ebola virus RNA in up to 24 samples without the need for amplification. In addition, Cas13 (combinatorial arrayed reactions for multiplexed evaluation of nucleic acids-Cas13, CARMEN-Cas13) combines microwell arrays and Cas13-based detection methods in a multi-channel detection method, which can be performed at one time Almost 5 000 tests were run simultaneously; in addition to virus detection, CARMEN-Cas13 can be used to distinguish all hemagglutinin (H1-H16) and neuraminidase (N1-N9) subtypes of influenza A virus. Combined with the amplification-free RNA detection method of the lipid bilayer microarray system developed earlier, Shinoda et al. established a Cas13-mediated RNA detection technology, which has been used to detect the 2019-nCoV.

Advantages and disadvantages of pathogen nucleic acid detection technology based on CRISPR-Cas system

Traditional pathogen detection methods include the cultivation of pathogenic microorganisms, antigen/antibody detection, PCR amplification detection, isothermal amplification technology, next-generation sequencing technology, etc. These technologies have their own characteristics. The most common method of detection. However, classical PCR detection requires special equipment and is used in a specific experimental environment. The high detection cost, long sample running cycle, and high requirements for personnel and equipment limit its application. Although many isothermal amplification methods reduce the requirements of hardware facilities and some of the detection time, their sensitivity and specificity are affected in order to achieve the requirements of simple and fast operation. Compared with these traditional methods, the CRISPR-Cas system has obvious advantages: high sensitivity, high specificity, low cost, low time consumption, no need for complex and expensive equipment, no need for personnel with a high level of expertise, and rapid detection in the field , and can be combined with different sample processing and amplification methods with unique Cas effector protein variants and innovative result readout methods, which further highlights its versatility in pathogen nucleic acid detection.