Chemically modified s-link based cas12a exponential amplification method

Abstract

The application discloses a chemical modification-based S chain and a Cas12a exponential amplification method, and belongs to the technical field of nucleic acid diagnosis.The chemical modification-based S chain comprises a thio-phosphoric acid modification region at both ends which can be complementary to S-crRNA and a middle trans-cleavage region, the trans-cleavage region uses a sequence of 5'-TTATT-3', and the thio-phosphoric acid modification region at both ends is subjected to thio-phosphoric acid modification every 0-2 bases apart, so as to resist Cas12a trans-cleavage.The application improves the existing CRISPR / Cas12a detection system, introduces a simple S chain, realizes the regulation of the Cas12a trans-cleavage region by means of thio-phosphoric acid modification, utilizes the characteristics of the split activator to activate Cas12a, and finally realizes the exponential amplification of CRISPR / Cas12a, so that the super-sensitive detection of low-abundance targets can be realized without additional target pre-amplification, the detection sensitivity can reach the fM level, and the operation is simple, without complex probe design, fast reaction and low cost.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid diagnostic technology, specifically to a chemically modified S-strand and Cas12a exponential amplification method. Background Technology

[0002] The CRISPR / Cas (Clustered Regularly Interspaced Short Palindromic Repeats / Cas) system was originally developed as an adaptive immune system in bacteria and archaea, and is now widely used in gene editing and diagnostics. This system mainly consists of a guide RNA (CRISPR-derived RNA) with a specific sequence and a Cas protease. When the crRNA binds to a complementary site to the target, it directly guides the Cas protease to cleave foreign nucleic acids, thereby achieving gene editing. It boasts advantages such as simplicity, reliability, high specificity, and sensitivity. CRISPR / Cas12a, also known as Cpf1, belongs to type V of the second class of CRISPR / Cas, possessing cis-cleavage activity and highly efficient non-specific trans-cleavage activity (kcat / KM>10). 6 s -1 M -1 By introducing a fluorescent and quencher-labeled single-stranded DNA signal probe, nucleic acid detection signal output can be achieved, thus making it widely used in the field of nucleic acid diagnostics. The activation modes of the Cas12a system are mainly divided into double-stranded DNA activation and single-stranded DNA activation. However, PAM sequence recognition is a prerequisite for double-stranded DNA activation, involving a series of complex processes such as double-stranded DNA unwinding, NTS strand (non-target strand) replacement, and R-loop formation. Compared to double-stranded DNA activators, single-stranded DNA activation is more direct and faster, not limited by the PAM sequence, and can directly induce a conformational change in Cas12a through base complementarity with crRNA, exposing the RuvC catalytic site, thereby undergoing trans-cleavage. Studies have also shown that splitting DNA can also activate Cas12a through synergistic effects.

[0003] However, single-stranded DNA activation-induced trans-splicing is a unidirectional process, resulting in linear signal amplification. This limits detection sensitivity to the pM level, making it difficult to meet the clinical needs for detecting low-abundance targets. Therefore, most early detection systems (such as DETECTR) relied on target pre-amplification to improve sensitivity. However, the introduction of pre-amplification procedures makes the detection system more complex and cumbersome. Consequently, many researchers, focusing on the characteristics of Cas12a itself, have attempted to cascade target activation and trans-splicing to achieve exponential signal amplification. For example, the Cas12a positive feedback signal cascade amplification strategy (CONAN), constructed in 2021, can achieve a detection sensitivity of aM under isothermal conditions. However, it also has certain drawbacks, such as the reaction initiation being highly dependent on changes in nucleic acid secondary structure, and the relatively complex probe design, hindering its widespread application.

[0004] Based on this, the present invention designs a chemically modified S-chain and Cas12a exponential amplification method to solve the above problems. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, this invention provides a chemically modified S-chain and Cas12a exponential amplification method. By introducing phosphate thiophosphate modification-mediated trans-cleavage into the Cas12a system, a simple and sensitive CRISPR-Cas exponential amplification method driven by a phosphate thiophosphate-modified cleavage activator is developed.

[0006] To achieve the above objectives, the present invention provides the following technical solution: The chemically modified S-strand contains phosphate-thiolated regions at both ends that are complementary to S-crRNA and a trans-cleavage region in the middle. The trans-cleavage region uses a 5'-TTATT-3' sequence. The phosphate-thiolated regions at both ends are modified with phosphate-thiolated every 0 to 2 bases to resist Cas12a trans-cleavage.

[0007] Furthermore, the sequence of the S chain is SEQ ID NO.1.

[0008] To better achieve the objectives of this invention, the present invention also provides the application of the chemically modified S-chain described above in the Cas12a exponential amplification reaction.

[0009] To better achieve the objectives of this invention, this invention also provides a Cas12a exponential amplification method based on the chemically modified S-chain, comprising the following steps: (1) Incubate the target, Cas12a / T-crRNA complex and S strand at 37°C for 5 min. The target will activate a small amount of Cas12a and cleave part of the S strand. (2) Add the Cas12a / S-crRNA complex corresponding to the S chain and FQ-ssDNA, so that the S chain product after cleavage will reactivate the remaining Cas12a in a fission activation manner. The large number of activated Cas12a cleave FQ-ssDNA and release a large amount of fluorescence.

[0010] Furthermore, the target sequence is SEQ ID NO.2.

[0011] Furthermore, the sequence of T-crRNA is SEQ ID NO.3.

[0012] Furthermore, the sequence of S-crRNA is SEQ ID NO.4.

[0013] Furthermore, the FQ-ssDNA sequence is: / i6FAMdT / TATT / iBHQ1dT / .

[0014] Compared with the prior art, the beneficial effects of this invention are as follows: This invention improves the existing CRISPR / Cas12a detection system by introducing a simple S chain and using thiophosphate modification to regulate the trans-cleavage region of Cas12a. By utilizing the cleavage activator to activate the characteristics of Cas12a, exponential amplification of CRISPR / Cas12a is finally achieved. No additional target pre-amplification is required to achieve ultrasensitive detection of low-abundance targets. The detection sensitivity can reach the fM level. Moreover, the operation is simple, no complex probe design is required, the reaction is fast, and the cost is low. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0016] Figure 1 This is a schematic diagram of the Cas12a exponential amplification method of the present invention; Figure 2 This experiment serves as a feasibility verification of the invention, demonstrating the effect of DNA strands modified with different thiophosphates on the ability to activate Cas12a.

[0017] Figure 3 The feasibility verification experiment of this invention demonstrates the ability of different thiophosphate modification methods to withstand Cas12a trans-shearing.

[0018] Figure 4 This experiment demonstrates the feasibility verification of the invention by implementing exponential signal amplification using an S-chain driven system.

[0019] Figure 5 This experiment serves as a verification experiment for the detection sensitivity of the present invention.

[0020] Figure 6 Design a route for the S-chain; Figure 7 This is a schematic diagram of the S-chain structure. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0022] Definitions of abbreviations and key terms: CRISPR / Cas12a: the acquired immune system in prokaryotes, now widely used in gene editing, nucleic acid diagnostics and other fields.

[0023] crRNA (CRISPR-derived RNA): An RNA molecule in the CRISPR / Cas system that primarily guides Cas proteins to locate and recognize target DNA, and to pair complementaryly with the target DNA.

[0024] NTS (non-target strand): The DNA strand in the target double-stranded DNA that is not complementary to crRNA.

[0025] Example 1: S-chain Design The design of the S-chain mainly includes phosphate-thiolated regions at both ends and a trans-cleavage region in the middle. Since Cas12a tends to cleave sequences rich in AT bases, and the cleavage product is usually 2-4 nt long, the trans-cleavage region is a 5'-TTATT-3' sequence. The phosphate-thiolated regions at both ends need to ensure that they do not affect the activation efficiency of Cas12a, while to the greatest extent possible to withstand the trans-cleavage of Cas12a, so that the activated Cas12a can effectively cleave the middle "protrusion" part. Therefore, a method of phosphate-thiolated modification every two bases is used, and finally, the Cas12a exponential amplification reaction based on phosphate-thiolated modification is constructed.

[0026] For example, the S chain sequence is: C*GCT*AGG*TAT*TTATTC*AGG*AGT*GC*G, where * represents thiophosphate modification; the S chain has a protrusion (TTATT) in the middle, and the 10 bases at both ends are modified with thiophosphate at intervals of 2 bases. The last three GCGs are prevented from being cleaved, so an additional thiophosphate modification is added.

[0027] Figure 2 The effects of DNA strands modified with different thiophosphates on their ability to activate Cas12a were investigated. "s" represents the thiophosphate-modified base, and "p" represents the spacer base. Complete thiophosphate modification of the sequence significantly inhibited Cas12a activation; however, thiophosphate modification with a 1-, 3-, or 5-base spacer resulted in Cas12a activation similar to that without thiophosphate modification. The concentrations of s20p0, s1p1, s1p3, s1p5, and s0p20 were 10 nM, the concentrations of Cas12a and S-crRNA were both 10 nM, and the concentration of FQ-ssDNA was 500 nM. The reaction was carried out at 37°C for 1 h. s20p0 represents all 20 bases modified with thiophosphate; s1p1 represents 20 bases modified with thiophosphate at intervals of 2 bases; s1p3 represents 20 bases modified with thiophosphate at intervals of 3 bases; s1p5 represents 20 bases modified with thiophosphate at intervals of 5 bases; s0p20 represents 20 bases not modified with thiophosphate.

[0028] Figure 3 In this study, PAGE was used to verify the varying degrees of resistance to Cas12a trans-cleavage under different phosphate-thiolated modifications. DNA strands with all phosphate-thiolated modifications exhibited the greatest resistance to trans-cleavage when used as trans-cleavage substrates. Resistance to trans-cleavage gradually decreased with increasing number of spacer bases until complete cleavage was achieved. The concentrations of s20p0, s1p1, s1p3, s1p5, and s0p20 were 2 μM, and the concentrations of the activation target and the corresponding Cas12a / crRNA complex were 1 nM. The reaction was carried out at 37 °C for 1 h.

[0029] Figure 4 In this study, the exponential signal amplification achieved by the S-chain-driven system was verified using endpoint fluorescence assay. Compared to Cas12a activation by the TS chain alone, the fluorescence signal intensity increased twofold upon the addition of the S chain, indicating that the S chain can be "trans-cleaved" by activated Cas12a, releasing the sequences at both ends, thereby co-activating Cas12a again in a cooperative manner, achieving signal cascading. The concentration of the S chain was 10 nM, the concentration of the TS chain was 1 nM, and the corresponding Cas / crRNA complex concentrations were both 10 nM; the reaction time was 30 min.

[0030] Figure 5 The lowest sensitivity can be 100 fM. The S chain concentration is 10 nM, and the corresponding Cas12a / S-crRNA and Cas12a / T-crRNA concentrations for the S and TS chains are 10 nM, respectively. The reaction is carried out at 37°C for 30 min.

[0031] This invention improves upon existing CRISPR / Cas12a detection systems by introducing a simple S-chain and using phosphate thioate modification to regulate the trans-cleavage region of Cas12a. By utilizing the cleavage activator to activate Cas12a, exponential amplification of CRISPR / Cas12a is achieved. This enables ultrasensitive detection of low-abundance targets without additional target pre-amplification, achieving a detection sensitivity down to the fM level. Furthermore, the system is simple to operate, requires no complex probe design, and features rapid reaction and low cost.

[0032] Example 2: A method for exponential amplification of Cas12a based on phosphate thiophosphate modification, comprising the following steps: (1) Incubate the target, Cas12a / T-crRNA complex and S strand at 37°C for 5 min. The target will activate a small amount of Cas12a and cleave part of the S strand. The Cas12a / T-crRNA complex was obtained by incubating Cas12a protein and T-crRNA at 37°C for 5 min; the Cas12a protein was purchased from NEB; and the T-crRNA was a biosynthesized RNA sequence.

[0033] The sequence of the S chain is as follows: C*GCT*AGG*TAT*TTATT C*AGG*AGT*GC*G.

[0034] The sequence of the target is: CCAAGTTTGAGACGGAACAGACTAGA.

[0035] The sequence of T-crRNA is: UAAUUUCUACUAAGUGUAGAUUCUAGUCUGUUCCGUCUCAA.

[0036] (2) Add the Cas12a / S-crRNA complex corresponding to the S chain and FQ-ssDNA, so that the S chain product after cleavage will reactivate the remaining Cas12a in a fission activation manner. The large number of activated Cas12a cleave FQ-ssDNA and release a large amount of fluorescence.

[0037] The Cas12a / S-crRNA complex was obtained by pre-incubating Cas12a protein and S-crRNA at 37°C for 5 min; the Cas12a protein was purchased from NEB; and the S-crRNA was a biosynthesized RNA sequence.

[0038] The sequence of S-crRNA is as follows: UAAUUUCUACUAAGUGUAGAUCGCACUCCUGAUACCUAGCGAAU.

[0039] The sequence of FQ-ssDNA is: / i6FAMdT / TATT / iBHQ1dT / .

[0040] Table 1 Sequence

[0041] This invention has a simple composition, mainly comprising a Cas12a / T-crRNA complex, a target, a Cas12a / S-crRNA complex, an S-strand, and FQ-ssDNA. The S-strand is a crucial component for achieving exponential CRISPR / Cas12a amplification. It has a simple structure, with 10 nt activators at both ends that are complementary to the S-crRNA. These activators are modified with thiophosphate to resist Cas12a trans-cleavage. The unmodified "TTATT" region in the middle serves as the trans-cleavage region. By adjusting the thiophosphate modification, activated Cas12a effectively cleaves the middle region, while the activator sequences at both ends remain unaffected. Therefore, when no target is present, although the S chain can bind to the Cas12a / S-crRNA complex, it prevents the correct conformational change of the Cas12a protein, i.e., it cannot activate Cas12a. However, when a target is present, the target binds to its corresponding Cas12a / T-crRNA and activates a small amount of Cas12a. The activated Cas12a acts on the trans-cleavage region of the S chain, and the resulting product reactivates other Cas12a in the manner of a fission activator, thus achieving exponential amplification of the signal.

[0042] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for exponential amplification of Cas12a based on chemically modified S-chains, characterized in that, Includes the following steps: (1) The target, Cas12a / T-crRNA complex and chemically modified S strand were incubated at 37°C for 5 min. The target activated a small amount of Cas12a and cleaved part of the chemically modified S strand. (2) Add the chemically modified S chain corresponding to the Cas12a / S-crRNA complex and FQ-ssDNA, so that the cleaved S chain product can reactivate the remaining Cas12a in a fission activation manner. The large amount of activated Cas12a cleaves FQ-ssDNA and releases a large amount of fluorescence. The chemically modified S-strand contains phosphate-thiolated regions at both ends that are complementary to S-crRNA and a trans-cleavage region in the middle. The trans-cleavage region uses a 5'-TTATT-3' sequence. The phosphate-thiolated regions at both ends are modified with a phosphate-thiolated molecule every 2 bases to resist Cas12a trans-cleavage. The sequence of the S-strand is SEQ ID NO.

1. The sequence of T-crRNA is SEQ ID NO.3; The sequence of S-crRNA is SEQ ID NO.4; The target sequence is SEQ ID NO.

2.

2. The Cas12a exponential amplification method according to claim 1, characterized in that, The FQ-ssDNA sequence is: / i6FAMdT / TATT / iBHQ1dT / .

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