General：DIRENJIE System
        Our detection system is divided into two modules, A and B. The user collects fingertip blood, drops it into the A tube of our supporting hardware (see hardware), and the microRNA to be tested enters the A module. If the microRNA target is present, it will hybridize to the probe. And duplex-specific nuclease (DSN) will cleave DNA in DNA-RNA hybrid duplex, re-releasing the microRNA and a complete sgRNA sequence will be released. The released microRNA can bind to a new probe for a new round of reaction. The released sgRNA sequence then self-folds to form a functional sgRNA.
        After a certain amount of reaction time, the plug on the lower part of tube A is opened and the reaction liquid flows into tube B (DSN is attached to the honeycomb bracket at the lower part of A and will not enter tube B).
        In the B system, the released sgRNA binds to the Cas12a protein, and then recruits the assistant DNA (later called aDNA), and Cas protein cleaves the aDNA with its cis-cleavage activity. After cleaving, Cas12a-sgRNA-aDNA forms a stable ternary complex, giving Cas12a trans-cleaving activity (non-specific ssDNA cleavage). After the trans-activity of Cas12a is activated, the DNA sequence of the circular DNA-RNA chimeric probe will be cleaved to release a new sgRNA sequence. The sgRNA sequence will self-fold to form a mature sgRNA and the newly generated sgRNA can activate a new Cas12a. Through this process, Cas12a amplifies exponentially. Activated Cas12a will also cleave the ssDNA between colloidal gold particles, so that the form of AuNPs will be changed from aggregation state to dispersion state, achieving the color transformation of the solution. We named this reaction system after Di Renjie, a very famous detective in ancient China.
        In brief, our system utilizes the double-strand (ds) DNA cleavage activity of DSN enzymes to achieve linear signal amplification and DNA trans-cleavage activity of Cas12a to achieve exponential signal amplification. A specially designed circular probe is synthesized to identify microRNA in system A, and its sgRNA sequence will be released to system B, achieving exponential amplification of the signal. Finally, colloidal gold particles (AuNPs) can convert specific microRNA in serum into visible light signals.
Single-stranded circular DNA-RAN chimeric probe
        The probe is the core part of our detection system. It identifies microRNAs in system A, and its sgRNA sequence is released in system B, achieving exponential amplification of the signal. This little probe connects system A and system B in series. Compared with the linear conformation, the circular probe has better stability, and it is not easy to bind Cas12a, which improves the detection efficiency of the system.

Our Design

        The sequence of our final version of the single-stranded circular DNA-RAN chimeric probe is shown below:         The DNA sequence in the probe is complementary to the microRNA sequence, and the RNA sequence is exactly the same as the sgRNA sequence we designed for Cas12a.
DSN system

DSN

        Duplex-specific nuclease (DSN)is a kind of protein enzyme that can effectively identify and cleave double-stranded DNA or DNA strand of DNA-RNA hybrid duplex, but doesn’t affect the single strand DNA or single/double strand RNA.[1]
        Substrate length requirements: minimum 9bp [2] or 10bp [1] DNA in dsDNA. Shorter double-stranded DNA will not be cut, and the effective cleaving length of a DNA-RNA hybrid duplex is 15bp [1]. The product cleaved by DSN enzyme consists mostly of short nucleotide chains and a small number of mononucleotides. When a DSN cleaves a partially double-stranded nucleic acid, there are 5-6 nucleotides at both ends of the double-stranded region that cannot be degraded.[3]

Our Design

        There are three parts in the DSN enzyme system: DSN, single-stranded circular DNA-RNA chimeric probe and small amounts of exonuclease I.         In this system, when target microRNA is present, it will pair with the DNA fragment of the circular probe to form a partial-duplex nucleic acid region. DSN cleaves the DNA strand in the partial-duplex nucleic acid region, but the DSN cleavage product has 5-6 nucleotides at both ends of the partial-duplex nucleic acid region that cannot be degraded. So, we introduced exonuclease I (only cleaves linear ssDNA) to further process the product, and produced sgRNA with optimal activity, which is introduced into the CRISPR-Cas12a system as a new signal.
CRISPR-Cas12a system

Cas12a

        The CRISPR-Cas system is an immune system naturally present in bacteria and archaea that is used to resist the invasion of foreign DNA. It protects bacteria from viral attacks by recognizing foreign DNA sequences and cleaving them into fragments. The core component of the CRISPR-Cas system is the Cas protein, which can recognize and cleave DNA. CRISPR-Cas systems become potential biosensing technologies for nucleic acid detection as a result of the recent discovery of the family of Cas12 nucleases with trans-cleavage activity.
        Cas12a is a Cas protein in the Cas12 family. It is an RNA-guided DNA endonuclease and belongs to the type 2 CRISPR-Cas system. Compared with Cas proteins in other CRISPR-Cas systems, Cas12a has some unique characteristics. First, Cas12a is able to maintain high cleavage activity over a wide temperature range, which allows it to function under various environmental conditions. Second, Cas12a has a smaller molecular weight, which makes it more convenient for in vivo delivery and application. Third, Cas12a also has high DNA cleavage efficiency and considerable non-specific cleavage tendency (trans-cleavage), which makes it suitable for precise nucleic acid detection.
        The activation mechanism of Cas12a is similar to other CRISPR-Cas systems. First, the Cas12a protein binds to sgRNA to form a Cas12a-sgRNA complex. This complex then recognizes the target DNA sequence and binds to a specific region of the target DNA sequence through base pairing. Once the Cas12a-sgRNA complex binds to the target DNA, the Cas12a protein cleaves the target DNA, resulting in a double-stranded break. At the same time, the trans-cleaving activity of Cas12a is activated, so that Cas12a can non-specifically cleave all single-stranded DNA.

Our Design

        In cell-free systems, Cas12a is also quite powerful.         In our DRJ system, Cas12a binds to sgRNA and cleaves assistant DNA in cis to obtain powerful trans-cleavage activity. Under room temperature and normal working conditions, the single-stranded circular DNA-RAN chimeric probes are efficiently cut to release new sgRNA. The newly generated sgRNA can activate a new Cas12a. Through this process, Cas12a is activated at an exponentially amplified rate. Activated Cas12a will also cleave ssDNA between colloidal gold particles, so that the form of AuNPs will be changed from aggregation state to dispersion state, achieving the color transformation of the solution. In this way, we achieve visualization of the signal.
Visualization system

Colloidal gold particles (AuNPs)

        Belonging to the heterogeneous system, Colloidal gold solution refers to the gold sol whose dispersed phase particle diameter is between 1-150 nm, and the color is orange-yellow to wine-red. The smallest colloidal gold (2 ~ 5nm) is orange-yellow, the medium-sized colloidal gold (10 ~ 20nm) is wine-red, and the larger particles of colloidal gold (30 ~ 80nm) are purplish red. Colloidal gold has a single light absorption peak (λ max) in the visible range, which is in the range of 510 ~ 550nm. With the size change of colloidal gold particles, λ max large colloidal gold is biased to long wavelength. On the contrary, λ max of small colloidal gold is biased to short wavelength.
        λ max of some colloidal gold listed in Table 1

Our Design

        In cell-free systems, Cas12a is also quite powerful.
        We chose colloidal gold particles with a diameter of 20nM and connected -SH to both ends of ssDNA. The modified ssDNA (sequence: SH-5'-AAAAAAAAAATAGCTTATCAGAAAAAAAAA-3'-SH) and dispersed colloidal gold particles were mixed and incubated. By forming Au-S bonds, the colloidal gold particles' state was changed from the dispersion state to the aggregation state. During the detection process, activated Cas12a will change the aggregation state of colloidal gold particles by cleaving ssDNA between colloidal gold particles, causing a series of color changes in the solution and converting nucleic acid signals into visible signals.
Reference
[1].   

Qiu X, Zhang H, Yu H, et al. Duplex-specific nuclease-mediated bioanalysis[J]. Trends in Biotechnology, 2015, 33(3): 180-188. DOI:10.1016/j.tibtech.2014.12.008

[2].   

Anisimova VE, Rebrikov DV, Shagin DA, et al. Isolation, characterization and molecular cloning of Duplex-Specific Nuclease from the hepatopancreas of the Kamchatka crab[J]. BMC Biochemistry, 2008, 9(1): 14. DOI:10.1186/1471-2091-9-14

[3].   

Zhao Y, Hoshiyama H, Shay JW, et al. Quantitative telomeric overhang determination using a double-strand specific nuclease[J]. Nucleic Acids Res, 2007, 36(3): e14. DOI:10.1093/nar/gkm1063