KR-20260067986-A - One-pot RPA/CRISPR-Cas12a assay with photo controlled aptamer-based inhibitor

Abstract

The present invention relates to a single-vessel analysis method for RPA/CRISSPR-CAS12A using a photo-modulated aptamer-based inhibitor. More specifically, the present invention can provide a nucleic acid detection method based on a recombinant polymerase amplification reaction and a CRISPR-Cas12a reaction within a single vessel, comprising the step of using a Cas12a inhibitory aptamer to which a photocleavable linker (PC) is introduced. Furthermore, the present invention provides a Cas12a inhibitory aptamer to which a photocleavable linker is introduced for detecting the p72 gene of African Swine Fever Virus (ASFV) based on a recombinant polymerase amplification reaction and a CRISPR-Cas12a reaction within a single vessel, and a method comprising the step of performing a recombinant polymerase amplification reaction and a CRISPR-Cas12a reaction within a single vessel using said Cas12a inhibitory aptamer. This concerns a gene detection method.

Inventors

• 정철희
• 조은영

Assignees

• 고려대학교 산학협력단

Dates

Publication Date

20260513

Application Date

20251023

Priority Date

20241105

Claims (13)

1. A step comprising using a Cas12a inhibitory aptamer into which a photocleavable linker (PC) has been introduced, A nucleic acid detection method based on Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a reactions in a single vessel.
2. In paragraph 1, A method characterized in that the above photodecomposition linker is cut by ultraviolet light.
3. In paragraph 1, A method characterized in that, during the recombinant enzyme polymerase amplification reaction, the Cas12a RNP (Ribonucleoprotein complex) binds to a Cas12a inhibitory aptamer into which the photolysis linker is introduced, thereby maintaining the trans-cleavage function of the Cas12a RNP in an inhibited state.
4. In paragraph 1, A method characterized by activating the transcleaving function of Cas12a RNP by cleaving a photodegradation linker within a Cas12a inhibitory aptamer by irradiating with ultraviolet light when the recombinant enzyme polymerase amplification reaction is completed.
5. In paragraph 1, The Cas12a inhibitory aptamer into which the above-mentioned photodegradation linker is introduced is, A flap region that binds complementarily to the seed region of the crRNA; and A method characterized by being composed of a stem-loop region that forms a hairpin structure and contains a PAM sequence at the end, which can bind to the PAM-binding domain of Cas12a.
6. In paragraph 5, A method characterized in that the flap region is composed of 4 to 10 nucleotide sequences that bind complementarily to the seed region of crRNA.
7. In paragraph 1, A method characterized by using the Cas12a inhibitor aptamer in a molar ratio of 1:1 to 1:2 to block Cas12a RNP.
8. In paragraph 5, The above photodegradation linker, with the flap-stem junction—which is the boundary between the flap and stem portions of the Cas12a inhibitory aptamer—as the 0 position, A method characterized by introducing at -4 position in the flap direction, -2 position in the flap direction, 0 position at the flap-stem joint, +5 position in the stem direction, +7 position in the stem direction, or +9 position in the stem direction.
9. In paragraph 2, A method characterized by irradiating the above ultraviolet light for 3 to 10 minutes.
10. As a Cas12a inhibitory aptamer with a photodegradable linker introduced for detecting the p72 gene of African Swine Fever Virus (ASFV) based on Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a reactions in a single vessel, The above Cas12a inhibitory aptamer is composed of a nucleotide sequence selected from the group consisting of SEQ ID NOs 1 to 8, and The above photodegradation linker is based on the flap-stem junction, which is the boundary between the flap and stem portions of the Cas12a inhibitory aptamer, as the 0 position, Characterized by being introduced at -4 position in the flap direction, -2 position in the flap direction, 0 position at the flap-stem joint, +5 position in the stem direction, +7 position in the stem direction, or +9 position in the stem direction. Cas12a inhibitor aptamer with a photodegradation linker introduced for detecting the p72 gene of African swine fever virus (ASFV).
11. A step comprising performing a recombinant enzyme polymerase amplification reaction and a CRISPR-Cas12a reaction in a single vessel using the Cas12a inhibitory aptamer of claim 10, Method for detecting the p72 gene of African swine fever virus.
12. In Paragraph 11, A method for detecting the p72 gene of the African swine fever virus, characterized in that the p72 gene of the African swine fever virus is composed of the nucleotide sequence of SEQ ID NO. 10.
13. In Paragraph 12, The above method is a method for detecting the p72 gene of African swine fever virus, characterized in that while a recombinant polymerase amplification reaction is performed in a single vessel, the trans-cleavage function of Cas12a RNP is inhibited by a Cas12a inhibitory aptamer, and after the completion of the recombinant polymerase amplification reaction, the linker present in the Cas12a inhibitory aptamer is cleaved by ultraviolet irradiation, thereby activating the trans-cleavage function of Cas12a RNP.

Description

One-pot RPA/CRISPR-Cas12a assay with photo-controlled aptamer-based inhibitor The present invention relates to a single-vessel RPA/CRISPR-Cas12a analysis method using a photo-modulated aptamer-based inhibitor, and more specifically, to a single-vessel RPA/CRISPR-Cas12a analysis method using a photo-modulated aptamer-based inhibitor that can detect target nucleic acids more effectively and sensitively by improving the problems that arise when performing Recombinase Polymerase Amplification (hereinafter RPA) and CRISPR-Cas12a reactions within a single vessel. Nucleic acid diagnostics is one of the most important tools for detecting pathogens due to its excellent sensitivity and specificity. In particular, CRISPR-Cas system-based nucleic acid diagnostics have recently been gaining prominence in molecular diagnostics due to their high sensitivity, specificity, robustness, and suitability for point-of-care testing. The CRISPR-Cas system originated from the adaptive immune systems of bacteria and archaea and was primarily used in fields such as gene editing due to its ability to cleave nucleic acids in a sequence-specific manner. However, following the discovery of trans-cleavage activity in type V Cas12 and type VI Cas13 proteins, the CRISPR-Cas system has expanded its applications to include molecular diagnostics. Unlike the cis-cleavage activity, which induces a single cleavage in a target nucleic acid complementary to the crRNA after forming a ribonucleoprotein complex (RNP) with CRISPR RNA (CRISPR RNA, hereinafter crRNA), the trans-cleavage activity is activated after recognizing a target nucleic acid and randomly cleaves surrounding single-stranded nucleic acids regardless of their sequence. Diagnostic methods such as SHERLOCK and DETECTR have been developed utilizing this. The principles of Sherlock and the detector are as follows. Viral nucleic acid is amplified from a sample using an isothermal amplification method called RPA. The amplified product is then mixed with a sample containing Cas12 or Cas13, appropriately designed crRNA, and FQ reporters—short single-stranded nucleic acids labeled with a fluorophore and a quencher. If viral nucleic acid is present, Cas12 RNPs or Cas13 RNPs recognize a specific sequence of the nucleic acid and activate a trans-cleavage function. This function causes surrounding FQ reporters to be cleaved, increasing the distance between the fluorophore and the quencher, thereby generating a fluorescent signal. If viral nucleic acid is absent, there is no nucleic acid to be amplified by RPA; consequently, there is no target sequence that Cas12 RNPs or Cas13 RNPs can recognize, and consequently, no fluorescent signal is generated. This method has the advantage of achieving high sensitivity at the atomolar level due to the fusion of nucleic acid amplification via RPA and the multi-turnover characteristics of the trans-cleavage functions of Cas12 and Cas13, and enabling sequence-specific signal induction by Cas proteins with high sequence-specificity. In addition, it has the advantage of eliminating the possibility of false-positives caused by non-specific amplification occurring in isothermal amplification. However, in the case of these early CRISPR diagnostic methods, nucleic acid amplification via RPA and the CRISPR reaction are carried out separately. Since dividing the reaction into two steps poses a risk of cross-contamination due to lid-opening and sample transfer, and the process is also complex, it is desirable to carry out the two reactions in a single container (one-pot). When nucleic acid amplification and CRISPR reactions are performed together within a single vessel, the amplification is generally carried out using the aforementioned RPA. RPA is an isothermal nucleic acid amplification method that utilizes recombinase to replace the denaturation process in the Polymerase Chain Reaction (PCR). It is known as an amplification method that is easy to apply to point-of-care diagnostics due to its reaction temperature of around 37°C. Since the optimal reaction temperature for Cas proteins is also around 37°C, compatibility between CRISPR reactions and RPA is possible. However, while performing the two reactions simultaneously within a single vessel offers the advantage of eliminating the possibility of cross-contamination and reducing the complexity of the workflow, it also has limitations. Since all components exist together in one container, the amplicon of RPA, which should act as the template for the next amplification, is continuously consumed by the cis-cleavage function of the Cas protein, and as the trans-cleavage function is activated, the primer of RPA, which is a single-stranded nucleic acid, is also consumed, so there is a problem that the efficiency of nucleic acid amplification decreases and sensitivity is reduced. Therefore, physical separation between RPA and CRISPR reactions is required, necessitating the development of improved technology to enable these r