CRISPR (clustered regularly interspaced short palindromic repeats) nucleic acid detection kit based on engineering crRNA scaffold and detection method thereof

Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats) nucleic acid detection kit based on an engineered crRNA scaffold and a detection method of the CRISPR nucleic acid detection kit, the CRISPR nucleic acid detection kit comprises the engineered crRNA scaffold, and the engineered crRNA scaffold is formed by connecting a light response group and a small fragment RNA to the 3 '-terminal of the crRNA scaffold. According to the detection kit and the detection method thereof disclosed by the invention, the engineered crRNA scaffold is formed by directly connecting the photoresponse group and the small-fragment RNA to the 3 '-tail end of the crRNA scaffold, so that large-batch production can be realized, screening and optimization are not needed, and the system design is greatly simplified. The engineered crRNA scaffold only contains one photoresponse group, so that the CRISPR-Cas12a function can be recovered under short-time illumination, and rapid and efficient nucleic acid detection can be realized. For different detection target objects, only the spacer matched with the detection target objects needs to be designed and synthesized, the flexibility is high, and the universality is good.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and relates to a CRISPR nucleic acid detection kit based on an engineered crRNA scaffold and a detection method thereof. Background Art

[0002] The CRISPR-Cas12a system can use a single CRISPR RNA (crRNA) to guide the Cas12a enzyme to recognize the T-rich protospacer adjacent motif (PAM) and recognize the target DNA, and then perform site-specific cleavage of the target DNA. In addition, the Cas12a enzyme activated by the target DNA can also non-specifically cleave single-stranded DNA (ssDNA). Due to the above characteristics, the CRISPR-Cas12a system can be coupled with a reporter co-labeled with a fluorophore and a quencher to produce a readable signal for biosensing.

[0003] Recent studies have shown that light-activated one-step CRISPR detection simplifies the analytical workflow while reducing the risk of cross-contamination and avoiding accidental CRISPR cleavage events, thereby achieving simple, efficient and ultrasensitive nucleic acid diagnostics. In order to achieve optical control of CRISPR-Cas12a function, researchers have designed and used chemically modified crRNA, including the use of photocleavable RNA protectors hybridized to crRNA and the embedding of masked nucleobases in the spacer region of crRNA. These methods help to achieve optical control of CRISPR-Cas12a function, but due to the sequence dependence of the target DNA, the number and position of the photocleavable groups need to be rigorously re-screened and optimized for different target DNAs. Although this problem can be solved by modifying the 2'-hydroxyl group of crRNA with a photosensitive small molecule, this method requires additional chemical synthesis and post-modification procedures. In addition, these strategies always require the re-synthesis or preparation of modified crRNA for each new target DNA, and therefore face the problems of complex design and poor universality.

[0004] Therefore, it is necessary to develop an optical control strategy for CRISPR-Cas12a function with simple design and strong universality to facilitate the development of a light-activated one-step CRISPR nucleic acid detection kit. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to develop an optical control strategy for CRISPR-Cas12a function with simple design and strong universality, apply it to light-activated one-step CRISPR nucleic acid detection, and provide related nucleic acid detection kits and nucleic acid detection methods to solve the problems of complex design and poor universality of the existing system.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a CRISPR nucleic acid detection kit based on an engineered crRNA scaffold, the components of which include an engineered crRNA scaffold, wherein the engineered crRNA scaffold is connected to a photoresponsive group and a small RNA fragment at the 3'-end of the crRNA scaffold.

[0008] In a preferred embodiment, the crRNA scaffold can be recognized by the Cas12a protein.

[0009] In a more preferred embodiment, the gene sequence of the crRNA scaffold includes but is not limited to SEQ ID No.1, SEQ ID No.5, and SEQ ID No.6.

[0010] In a preferred embodiment, the photoresponsive group adopts existing mature technology, such as PC-linker.

[0011] In a preferred embodiment, the length of the small RNA fragment is not less than 15 nt;

[0012] The small RNA fragment allows self-folding to form a secondary structure;

[0013] The number of each base of A, U, G and C in the small RNA fragment is at least 1.

[0014] In a preferred embodiment, the components of the CRISPR nucleic acid detection kit further include: Cas12a protein, spacer, reaction buffer, fluorescent probe, enzyme-free water and isothermal amplification system.

[0015] In a more preferred embodiment, the Cas12a protein is a class II type VA Cas protein, comprising one or more of the following proteins:

[0016] LbCas12a, AsCas12a, FnCas12a, ErCas12a, AbCas12a, ArCas12a, As2Cas12a, BbCas12a, BfCas12a, BoCa s12a, BsCas12a, CMaCas12a, CMtCas12a, CRbCas12a, CPbCas12a, EsCas12a, FbCas12a, HkCas12a, Lb4Ca s12a, Lb5Cas12a, LpCas12a, MaCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, MlCas12a, Pb2Cas12a, PdCas 12a, PgCas12a, PrCas12a, PxCas12a, PcCas12a, PbCas12a, Pb2Cas12a, PgbCas12a, SaCas12a, TsCas12a.

[0017] In a more preferred embodiment, the reaction buffer comprises: 10-100 mM Tris-HCl, 10-100 mM NaCl, 10-100 mM MgCl2, 1-100 mM DTT, and 1-10% by volume PEG-200.

[0018] In a more preferred embodiment, the fluorescent probe is modified with a fluorescent group and a quenching group, and the nucleotide sequence of the fluorescent probe contains at least one base among A, T, C, and G;

[0019] The length of the fluorescent probe is 5 to 40 nt;

[0020] The fluorescent probe is allowed to self-fold to form a secondary structure in which the fluorescent group and the quencher group are adjacent.

[0021] In a more preferred embodiment, the isothermal amplification system comprises recombinase polymerase amplification, recombinase-assisted amplification, helicase-dependent DNA isothermal amplification, and helicase-mediated polymerase chain reaction;

[0022] The isothermal amplification reagent used contains the enzymes, ions and primers required for amplification.

[0023] Among them, the small RNA fragment is used to prevent the spacer from entering the specific domain of the Cas12a protein to silence the enzymatic activity of the Cas12a protein; at the same time, the photoresponsive group has the characteristic of light-breaking bonds, which breaks after light excitation, so that the small RNA fragment is separated from the crRNA scaffold, and the spacer re-enters the specific domain of the Cas12a protein, thereby activating the CRISPR-Cas system.

[0024] The present invention also provides a CRISPR nucleic acid detection method based on an engineered crRNA scaffold, comprising the following steps:

[0025] (1) mixing the components in the nucleic acid detection kit to obtain a premixed reaction solution;

[0026] (2) mixing the sample to be tested with the premixed reaction solution and performing isothermal amplification;

[0027] (3) After the isothermal amplification is completed, UV illumination is performed, and finally the fluorescence signal is detected, and qualitative or quantitative analysis is performed based on the fluorescence signal value.

[0028] Furthermore, in step (2), the reaction temperature of the isothermal amplification is 20-50° C., and the reaction time is at least 5 minutes.

[0029] Furthermore, in step (3), the wavelength of the UV light source is 300nm to 420nm, and the illumination duration is at least 5s. During UV illumination, the wavelength of light is related to the characteristics of the photoresponsive group. When the wavelength of the light source is 365nm, the photoresponsive group of the present invention reacts most sensitively.

[0030] The mechanism of the present invention is as follows: the crRNA scaffold and the small RNA fragment use the photoresponsive group as the connection carrier, can be mass-produced, and do not need to be screened and optimized, which greatly simplifies the system design; because the system involves only one photoresponsive group, it can ensure that the CRISPR-Cas12a function is restored under short-term illumination, so that targets as low as 1 copy / μL (such as ASFV DNA and SARS-CoV-2 RNA) can be effectively detected, which is more than two orders of magnitude higher than the detection capacity of conventional test kits, with high detection efficiency, rapid reaction, sensitivity and accuracy; only spacers need to be designed and synthesized for different detection targets, with high flexibility and good versatility.

[0031] Beneficial technical effects of the present invention:

[0032] (1) The detection kit and detection method of the present invention, wherein the engineered crRNA scaffold is a photoresponsive group and a small RNA fragment directly connected to the 3'-end of the crRNA scaffold, can be mass-produced without the need for screening and optimization, and greatly simplifies the system design.

[0033] (2) The engineered crRNA scaffold contains only one photoresponsive group, which can ensure that the CRISPR-Cas12a function is restored under short-term illumination, enabling rapid and efficient nucleic acid detection.

[0034] (3) For different detection targets, only the matching spacer needs to be designed and synthesized, which has high flexibility and good versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a one-step CRISPR nucleic acid detection principle based on light activation of engineered crRNA scaffold;

[0036] Figure 2 This is a comparative analysis result of the CRISPR system activity before and after illumination of the present invention;

[0037] Figure 3 This is a comparative analysis result of the activity of the CRISPR system based on different crRNA scaffolds and different Cas proteins before and after illumination in the present invention;

[0038] Figure 4 This is a comparative analysis result diagram of ASFV DNA detection by the light-activated one-step CRISPR nucleic acid detection system of the present invention and the conventional one-step CRISPR nucleic acid detection system;

[0039] Figure 5 This is a comparative analysis result diagram of SARS-CoV-2 RNA detection by the light-activated one-step CRISPR nucleic acid detection system of the present invention and the conventional one-step CRISPR nucleic acid detection system;

[0040] Figure 6 This is a graph showing the results of clinical HPV sample detection using the light-activated one-step CRISPR nucleic acid detection system of the present invention. DETAILED DESCRIPTION

[0041] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0042] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0043] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market.

[0044] The principle of light-activated one-step CRISPR nucleic acid detection of the engineered crRNA scaffold of the present invention is shown in Figure 1 .Depend on Figure 1It can be seen that the present invention connects the crRNA scaffold and the small fragment RNA into one by PC-linker, and constructs a CRISPR-Cas system with light-activated properties. The presence of small fragment RNA hinders the action of spacer and Cas protein and target nucleic acid, thereby temporarily suspending the progress of CRISPR-Cas detection, and the activity of the CRISPR-Cas system can be reactivated by simple and convenient illumination operation. In this way, the CRISPR-Cas system with light-activated properties is integrated into the nucleic acid amplification system, which can achieve the purpose of separating nucleic acid amplification and CRISPR-Cas detection in the time dimension, and avoid the amplification inhibition caused by the cleavage activity of the CRISPR system.

[0045] The present invention is further described below by way of examples and drawings.

[0046] Example 1

[0047] Verify the activity of the CRISPR system before and after illumination

[0048] (1) Preparation of target dsDNA: A 100 μL reaction system contained 10 μM TS, 10 μM NTS, and 1×Cas12a reaction buffer (10 mM Tris-HCl, pH 8.9, 10 mM NaCl, 15 mM MgCl2, 1 mM DTT, 5% PEG-200);

[0049] (2) The reaction system was incubated at 95°C for 5 min, and then the temperature was gradually decreased at 2°C / min until the reaction temperature dropped to 25°C. The resulting product was stored at -80°C for subsequent use;

[0050] (3) preparing two reaction premixes, each of which contains LbCas12a protein, engineered crRNA scaffold, spacer, 1× reaction buffer, fluorescent probe, and enzyme-free water;

[0051] (4) The target dsDNA was added to the two premixed reaction solutions, one of which was not exposed to ultraviolet light, and the other was exposed to ultraviolet light for 30 seconds, and then incubated at 37°C for 60 minutes. After the reaction was completed, the fluorescence signal value was measured. The result was as follows: Figure 2 shown.

[0052] (5) The positive control was set as follows: the engineered crRNA scaffold was replaced with the crRNA scaffold; the negative control was set as follows: the target dsDNA was replaced with enzyme-free water.

[0053] Depend on Figure 2It shows that without UV illumination, the presence of target DNA cannot cause a significant change in the fluorescence signal, indicating that the engineering of crRNA scaffold can effectively inhibit the activity of the CRISPR system; after UV illumination, the activity of the CRISPR system is restored due to the departure of small RNA fragments, generating a fluorescence signal equivalent to that when the crRNA scaffold is used directly, indicating that light can effectively restore the activity of the CRISPR system.

[0054] In this experiment, the relevant information of the nucleic acid sequences used is shown in Table 1:

[0055] Table 1: Sequence information used in Example 1

[0056]

[0057] Example 2

[0058] Verify the activity changes of CRISPR systems based on different crRNA scaffolds and different Cas proteins before and after illumination

[0059] (1) preparing target dsDNA according to the steps in Example 1;

[0060] (2) preparing two reaction premixes, each of which contains AsCas12a protein (or ErCas12a), engineered crRNA scaffold 2, spacer, 1× reaction buffer, fluorescent probe, and enzyme-free water;

[0061] (3) preparing two reaction premixes, each of which contains FnCas12a protein, engineered crRNA scaffold 3, spacer, 1× reaction buffer, fluorescent probe, and enzyme-free water;

[0062] (4) The target dsDNA was added to the two premixed reaction solutions, one of which was not exposed to ultraviolet light, and the other was exposed to ultraviolet light for 30 seconds, and then incubated at 37°C for 60 minutes. After the reaction was completed, the fluorescence signal value was measured. The result was as follows: Figure 2 shown.

[0063] (5) The positive control was set as follows: replacing engineered crRNA scaffold 2 with crRNA scaffold 2, and replacing engineered crRNA scaffold 3 with crRNA scaffold 3; the negative control was set as follows: replacing target dsDNA with enzyme-free water.

[0064] Depend on Figure 3It shows that without UV illumination, the presence of target DNA cannot cause significant changes in the fluorescence signal, indicating that the engineering of crRNA scaffold can effectively inhibit the activity of the CRISPR system; after UV illumination, the activity of the CRISPR system is restored due to the departure of small RNA fragments, generating a fluorescence signal equivalent to that when crRNA scaffolds 2 and 3 are used directly, indicating that light can effectively restore the activity of the CRISPR system.

[0065] In this experiment, the relevant information of the nucleic acid sequences used is shown in Table 3:

[0066] Table 2: Sequence information used in Example 2

[0067]

[0068] Example 3

[0069] Verify the feasibility of a light-activated one-step CRISPR nucleic acid detection method based on an engineered crRNA scaffold (with a one-step CRISPR nucleic acid detection system based on a crRNA scaffold as a control)

[0070] (1) preparing a photoactivated one-step CRISPR detection reaction premix, the premix comprising Cas12a protein, engineered crRNA scaffold, spacer, 1× reaction buffer, fluorescent probe, enzyme-free water, and isothermal amplification system;

[0071] (2) Add different concentrations of target nucleic acid (1000 copies / μL, 100 copies / μL, 10 copies / μL, 1 copy / μL and 0.1 copy / μL) to the premixed reaction solution and react at 37°C for 20 minutes. Then, irradiate the premixed reaction solution with UV light for 30 seconds. Finally, the reaction solution was incubated at 37°C for 70 minutes, and the fluorescence signal value of the reaction system per minute was read using a real-time fluorescence instrument. In this experiment, recombinase polymerase amplification was used for DNA target amplification; reverse transcription-recombinase polymerase amplification was used for RNA target amplification.

[0072] (3) preparing a conventional one-step CRISPR detection reaction premix, which contains Cas12a protein, crRNA scaffold, spacer, 1× reaction buffer, fluorescent probe, enzyme-free water, and isothermal amplification system;

[0073] (3) Add different concentrations of target nucleic acid (1000 copies / μL, 100 copies / μL, 10 copies / μL, 1 copy / μL and 0.1 copy / μL) to the premixed reaction solution and incubate at 37°C for 90 min, and use a real-time fluorescence instrument to read the fluorescence signal value of the reaction system every minute; in this experiment, recombinase polymerase amplification was used for DNA target amplification; reverse transcription-recombinase polymerase amplification was used for RNA target amplification;

[0074] (4) Negative control was set up as follows: enzyme-free water was used instead of target nucleic acid.

[0075] The results are as follows Figure 4-5 As shown in the figure, it can be seen that the light-activated one-step CRISPR nucleic acid detection system based on the engineered crRNA scaffold can effectively detect ASFV DNA and the O gene of SARS-CoV-2RNA as low as 1 copy / μL, while the conventional one-tube CRISPR nucleic acid detection system can only detect 100 copies / μL of ASFV DNA and SARS-CoV-2RNA. In comparison, the detection capability of the method of the present invention is two orders of magnitude higher than that of the conventional method.

[0076] In this experiment, the sequence information used is shown in Table 3:

[0077] Table 3: Sequence information used in Example 3

[0078]

[0079]

[0080] Example 3

[0081] Verify the feasibility of a light-activated one-step CRISPR nucleic acid detection system based on an engineered crRNA scaffold for HPV clinical sample detection

[0082] (1) preparing a premixed reaction solution according to step 1 of Example 2;

[0083] (2) The positive and negative samples determined by qPCR were added to the premixed reaction solution and incubated at 37°C for 20 minutes. Subsequently, the premixed reaction solution was irradiated with UV light for 30 seconds. Finally, the reaction solution was incubated at 37°C for 70 minutes, and the fluorescence signal value of the reaction system per minute was read using a real-time fluorescence instrument. In this experiment, recombinase polymerase amplification was used for sample amplification;

[0084] The results are as follows Figure 6 As shown in the figure, it can be seen that this method can effectively distinguish clinical HPV-16 positive samples and negative samples.

[0085] In this experiment, the sequence information used is shown in Table 4:

[0086] Table 4: Sequence information used in Example 4

[0087]

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of implementation of the present invention. If the present invention is modified or replaced by equivalents without departing from the spirit and scope of the present invention, it should be included in the protection scope of the claims of the present invention.

Claims

1. A CRISPR nucleic acid detection kit based on an engineered crRNA scaffold, characterized in that: Its components include an engineered crRNA scaffold, wherein the engineered crRNA scaffold is a 3'-end of which a light response group and a small RNA fragment are connected.

2. A CRISPR nucleic acid detection kit based on an engineered crRNA scaffold according to claim 1, characterized in that, The crRNA scaffold can be recognized by the Cas12a protein.

3. A CRISPR nucleic acid detection kit based on an engineered crRNA scaffold according to claim 1, characterized in that: The gene sequence of the crRNA scaffold includes but is not limited to SEQ ID No.1, SEQ ID No.5, and SEQ ID No.

6.

4. A CRISPR nucleic acid detection kit based on an engineered crRNA scaffold according to claim 1, characterized in that The length of the small RNA fragment is not less than 15 nt; The small RNA fragment allows self-folding to form a secondary structure; The number of each base of A, U, G and C in the small RNA fragment is at least 1.

5. A CRISPR nucleic acid detection kit based on an engineered crRNA scaffold according to claim 1, characterized in that: The components of the CRISPR nucleic acid detection kit also include: Cas12a protein, spacer, reaction buffer, fluorescent probe, enzyme-free water and isothermal amplification system.

6. A CRISPR nucleic acid detection kit based on an engineered crRNA scaffold according to claim 5, characterized in that: The Cas12a protein is a class II type VA Cas protein, including LbCas12a, AsCas12a, FnCas12a, ErCas12a, AbCas12a, ArCas12a, As2Cas12a, BbCas12a, BfCas12a, BoCas12a, BsCas12a, CMaCas12a, CMtCas12a, CRbCas12a, CPbCas12a, EsCas12a, FbCas12a, HkCas 12a, Lb4Cas12a, Lb5Cas12a, LpCas12a, MaCas12a, MbCas12a, Mb2Cas12a, Mb3Cas12a, MlCas12a, Pb2Cas12a, PdCas12a, PgCas12a, PrCas12a, PxCas12a, PcCas12a, PbCas12a, Pb2Cas12a, PgbCas12a, SaCas12a, TsCas12a or more.

7. A CRISPR nucleic acid detection kit based on an engineered crRNA scaffold according to claim 5, characterized in that: The components of the reaction buffer include: 10-100 mM Tris-HCl, 10-100 mM NaCl, 10-100 mM MgCl2, 1-100 mM DTT, and 1-10% volume percentage PEG-200.

8. A CRISPR nucleic acid detection kit based on an engineered crRNA scaffold according to claim 5, characterized in that: The fluorescent probe is modified with a fluorescent group and a quenching group, and the nucleotide sequence of the fluorescent probe contains at least one base among A, T, C, and G; The length of the fluorescent probe is 5 to 40 nt; The fluorescent probe is allowed to self-fold to form a secondary structure in which the fluorescent group and the quencher group are adjacent.

9. A CRISPR nucleic acid detection method based on an engineered crRNA scaffold, characterized in that: The following steps are involved: (1) mixing the components in the nucleic acid detection kit to obtain a premixed reaction solution; (2) mixing the sample to be tested with the premixed reaction solution and performing isothermal amplification; (3) After the isothermal amplification is completed, UV illumination is performed, and finally the fluorescence signal is detected, and qualitative or quantitative analysis is performed based on the fluorescence signal value.

10. A CRISPR nucleic acid detection method based on an engineered crRNA scaffold according to claim 9, characterized in that: In step (2), the reaction temperature of the isothermal amplification is 20-50° C., and the reaction time is at least 5 min; In step (3), the wavelength of the UV light source is 300nm to 420nm, and the illumination time is at least 5s.

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