KR20210087363A - Method for Detecting Nucleic Acid with High Sensitivity using CRISPR/Cas Assisted Lower temperature PCR - Google Patents

Abstract

The present invention relates to a method for detecting a nucleic acid with high sensitivity and specificity within a short time in which a target nucleic acid is reacted with a guide RNA specific for a target nucleic acid and a CAS protein to generate a cleaved target nucleic acid fragment having a low heat denaturation temperature, and the target nucleic acid fragment is subjected to a polymerase chain reaction in a low temperature heat denaturation step. In the case of using a PCR method according to the present invention, even in a sample containing a high concentration of substrate nucleic acid, there is an advantage of quickly detecting the nucleic acid in the field with high sensitivity and specificity like digital PCR without being affected by the substrate nucleic acid.

Description

High-sensitivity nucleic acid detection method using CAL PCR {Method for Detecting Nucleic Acid with High Sensitivity using CRISPR/Cas Assisted Lower temperature PCR}

The present invention relates to a method for high-sensitivity nucleic acid detection using CRISPR/Cas supported low-temperature PCR, and more particularly, a cleaved target nucleic acid fragment having a low heat denaturation temperature by reacting a target nucleic acid with a guide RNA and a CAS protein specific for the target nucleic acid. It relates to a method for detecting nucleic acids with high sensitivity and specificity within a short time by generating a polymerase chain reaction of the target nucleic acid fragment in a low temperature heat denaturation step.

Polymerase chain reaction (PCR) is a core technology for molecular diagnosis, and quantitative analysis is possible with real-time quantitative PCR, and various multiplex PCR (multiplex PCR) techniques have been developed. It has developed into an important technique for diagnostics (syndromic nucleic acid-based POCT diagnostics). However, PCR-based methods currently used have problems in that sensitivity and specificity are low in the case of a sample containing a high concentration of matrix nucleic acid. For example, in the case of whole blood used for point-of-care or sepsis diagnosis, it contains a high concentration of genomic DNA extracted from blood cells compared to serum or plasma and amplifies a very small amount of pathogens. There is a problem that non-specific amplification occurs when doing this, and sensitivity and specificity are lowered. Although digital PCR technology has been developed to solve these problems, expensive equipment and reagents are required, and there is a problem in processing a large number of samples. In addition, digital PCR takes a lot of time for analysis and has limitations in that it cannot be used for on-site diagnosis because fully automated equipment from nucleic acid extraction has not been developed. Therefore, there is an urgent need to develop a PCR method, particularly a real-time quantitative PCR method, that can detect a trace amount of a target with high sensitivity and specificity even in a sample containing a high concentration of substrate nucleic acid.

The CRISPR/Cas9 system is an immune system found in bacteria and acts as a defense mechanism against phages. This is an RNA-induced gene scissors that recognizes the target base of the target DNA by single-stranded guide RNA (sgRNA) and causes DNA double-strand break (DSB) by the nuclease activity contained in Cas9 protein. to be. This system has the advantage of being able to target and edit any DNA site through sgRNA sequence change only if there is only sgRNA that recognizes 20 base sequences and a protosapcer adjacent motif (PAM) sequence that recognizes Cas9 protein. The CRISPR/CAS12 (cpf1) system also differs only in the Pam sequence, and DNA double-strand break (DSB) is possible by nuclease activity.

Recently, research using the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas system has been actively conducted in the field of molecular diagnostics, and it is expected that it will play an important role in developing next-generation molecular diagnostics. The first use of CRISPR gene scissors for molecular diagnosis was the Sherlock (Specific High-sensitivity Enzymatic Reporter unlocking) technology announced in 2017 by Professor Feng Zang of MIT in the US. Since then, CRISPR gene scissors-based molecular diagnostic technology has been developed rapidly, leading to the development of HOLMES, HUDSON-SHERLOCK, and DETECTR.

These techniques can be broadly divided into two types, both of which are methods for obtaining a signal after first amplifying a sample with PCR or isothermal amplification methods RCA, NASBA, and LAMP. The first type is a method of detecting using a fluorescent probe using the non-specific nucleolytic activity of PCR CRISPR/Cas13 (SHELOCK, HUDSON, etc.), and the second type is dCAS9 with the nucleolytic activity of Cas9 protein removed. It can be divided into a form of amplifying a signal by linking an enzyme to the bound nucleic acid using the sequence-specific binding of

In general, when the target DNA to be detected is mixed in a small amount with a large amount of non-target DNA, it is often difficult to accurately detect the target DNA due to the non-specific competitive reaction of the non-target DNA. For example, when it is necessary to detect bacteria in whole blood, such as in sepsis, the amount of DNA contained in 1 ml of blood is 30-40 ug/ml and the amount of RNA is about 1/10, 1-5 ug/ml. ml, so it is very difficult to accurately detect one to several tens of small bacteria or viruses contained therein. Since genomic DNA contained in a large amount in blood causes non-specific amplification by non-specific priming of the detection primer, it is difficult to selectively expand and detect target bacteria or viruses. This is a problem that frequently occurs in detecting specific bacteria or viruses in cell-derived samples containing a lot of DNA as well as sepsis.

Meanwhile, the demand for POCT diagnosis, which can be quickly diagnosed in the field using molecular diagnostic methods, is increasing. In this case, it is necessary to additionally increase the time for the initial thermal denaturation step at 95° C. for the subsequent amplification to occur. In this case, the test time increases according to the additionally increased initial heat denaturation step. In the subsequent steps of hybridization and polymerization (annealing & polymerization), the shorter the temperature difference from the previous heat denaturation step, the shorter the time required for temperature cycling and energy savings. However, in general PCR, the temperature of the heat denaturation step is 95 ℃ proceeds.

As a result of the present inventors' earnest efforts to find a more accurate and rapid PCR-based molecular diagnostic method in the environment for sequence-specific hydrolysis of CRISPR/Cas protein, a plurality of samples in which trace amounts of target nucleic acids and excess non-target nucleic acids are mixed In the case of using the PCR method introducing the CRISPR/Cas system with guide RNA, the present invention confirmed that it is possible to detect target nucleic acids with high sensitivity and specificity, as well as shorten the PCR reaction time required for on-site molecular diagnosis. has been completed

It is an object of the present invention to provide a method, composition, and kit for diagnosing a disease that can detect a target nucleic acid in a short time with high sensitivity and specificity in a sample in which a trace amount of a target nucleic acid and an excessive amount of a non-target nucleic acid are mixed.

In order to achieve the above object, the present invention comprises the steps of reacting a target nucleic acid-specific guide RNA and a CAS protein with a target nucleic acid to generate a cleaved target nucleic acid fragment; and polymerase chain reaction (PCR) of the target nucleic acid fragment. It provides a method for detecting a target nucleic acid comprising a.

The present invention also provides a guide RNA specific for a target nucleic acid or a DNA encoding the guide RNA; a Cas protein or a nucleic acid encoding the Cas protein; DNA polymerase; a pair of primers for amplification of a target nucleic acid; dNTPs; And it provides a composition for detecting a nucleic acid comprising ^{Mg 2 +.}

The present invention also provides a kit for diagnosing a disease comprising the composition.

In the case of using the CAL PCR (CRISPR/Cas Assisted Lower temperature PCR) method according to the present invention, even in a sample containing a high concentration of substrate nucleic acid, it is not affected by the substrate nucleic acid and quickly in the field with high sensitivity and specificity like digital PCR. It has the advantage of being able to detect it.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

The present invention selectively cuts a target nucleic acid using two or more single-stranded guide RNAs and a Cas protein designed so that the denaturation temperature of the target nucleic acid fragment to be amplified is 90° C. or less, and the resulting target nucleic acid fragment is cooled at low temperature. It relates to a PCR method for amplifying in

In general, since the double helix structure of substrate-derived DNA is released at 95°C, non-specific amplification does not occur because the primer cannot bind to the substrate-derived DNA in PCR performed at a low denaturation temperature of 92°C or less. On the other hand, the target DNA fragment cut by CRISPR/Cas according to the present invention can be hybridized with the primer and amplified through the PCR reaction because the double helix is released in the PCR reaction proceeding at a low heat denaturation temperature. The present invention is a CAL (CRISPR/Cas Assisted Low temperature)-PCR reaction in which a CRISPR/Cas system that specifically cuts target DNA to have a low heat denaturation temperature and a PCR reaction proceeding at a low heat denaturation temperature are combined.

The present invention can be used to detect target DNA or RNA present in a trace amount in a sample containing an excess of non-target DNA or RNA. In particular, in the case of an RNA sample, cDNA generated through reverse transcription PCR (RT-PCR) may be subjected to a CRISPR/Cas Assisted Low temperature (CAL)-RT PCR reaction by applying the same principle as described above. In addition, after cleaving RNA using CRISPR/Cas13, it can be amplified using RT-PCR at a low heat denaturation temperature. By combining the above methods, it is possible to simultaneously and highly sensitively amplify trace amounts of target DNA and target RNA in a sample containing an excess of non-target nucleic acid.

In the present invention, the PCR cycle time can be significantly reduced by generating a fragment of the target nucleic acid to have a low heat denaturation temperature. For example, when the thermal denaturation temperature is lowered to 75 °C and the hybridization temperature is 55 °C in the present invention, compared to the reaction with the thermal denaturation temperature of 95 °C used in general PCR and the hybridization temperature of 55 °C, the reaction You can cut the time by 1/2. This has the advantage of enabling rapid POC diagnosis, and results can be quickly confirmed in field-type molecular diagnosis. In addition, there is a problem in that the activity of the heat-resistant DNA polymerase is rapidly reduced at a temperature of 95° C. or higher. By inducing a low heat denaturation temperature, the PCR reaction can be performed without decreasing the activity of the enzyme.

Accordingly, the present invention comprises the steps of reacting a target nucleic acid-specific guide RNA and a CAS protein with a target nucleic acid to generate a cleaved target nucleic acid fragment; and polymerase chain reaction (PCR) of the target nucleic acid fragment.

In the present invention, the guide RNA may be designed so that the thermal denaturation temperature of the cleaved target nucleic acid is 65 to 94°C. As an aspect of the present invention, the guide RNA may be designed so that the heat denaturation temperature of the cleaved target nucleic acid is 90° C. or less, preferably designed to be 70 to 90° C. , but is not limited thereto.

In addition, the temperature of the thermal denaturation step in the polymerase chain reaction is preferably the same as or higher than the thermal denaturation temperature of the target nucleic acid fragment, but preferably does not exceed 95°C. The temperature of the heat denaturation step in the polymerase chain reaction may be 65 to 94 ℃. As an aspect of the present invention, the temperature of the heat denaturation step in the polymerase chain reaction may be characterized in that it is 92° C. or less, preferably 70 to 92° C., but is not limited thereto.

In the present invention, the thermal denaturation temperature of the cleaved target nucleic acid may be designed to be lower than the thermal denaturation temperature of the non-target nucleic acid, which can be achieved by cutting the target nucleic acid with guide RNA and shortening the length. have. The length of the cleaved target nucleic acid may be about 100 bp, preferably 50 to 150 bp, more preferably 70 to 120 bp, but is not limited thereto.

In the present invention, the guide RNA may be characterized as two or more guide RNA pairs, and the guide RNA may be a single-stranded guide RNA.

The present invention also provides a guide RNA specific for a target nucleic acid or a DNA encoding the guide RNA; a Cas protein or a nucleic acid encoding the Cas protein; DNA polymerase; a pair of primers for amplification of a target nucleic acid; dNTPs; And Mg ^{2 + It} relates to a composition for detecting a nucleic acid comprising.

In the present invention, the guide RNA may be designed so that the thermal denaturation temperature of the cleaved target nucleic acid is 90° C. or less.

The composition may further include a fluorescent probe, reverse transcriptase, and/or a stabilizing agent, if necessary, and the stabilizing agent is sorbitol, mannitol, galactitol, sucrose. , trehalose (trehalose) and dextran (dextran) may be characterized as a carbohydrate selected from the group consisting of, but is not limited thereto.

In the present invention, the "nucleic acid" is used in the meaning including, without limitation, a template nucleic acid to be amplified, DNA, RNA, a hybrid of DNA and RNA, a mixture thereof, a nucleic acid extract, or a nucleic acid sample.

In addition, the present invention can be used to detect a mutant gene present in a trace amount in a sample by designing the CRISPR sgRNA to make a DNA fragment having a low transformation temperature, including selectively cleaving a mutant cancer gene site. In addition, it can be used to detect even trace amounts of cancer genes by translocation, such as the Philadelphia genome of chronic myeloid leukemia.

In addition, the present invention can selectively amplify a trace amount of target RNA when an excess of non-target RNA is present.

In other words, it is possible to perform field-type multimolecular diagnostics capable of simultaneously detecting both target DNA and target RNA present in trace amounts in a sample containing a high concentration of non-target nucleic acid with high sensitivity.

Accordingly, the present invention relates to a kit for diagnosing a disease comprising the composition in another aspect.

In the present invention, the disease diagnosis kit may be a cancer diagnosis kit, and the target nucleic acid may include a cancer-related gene mutation.

As used herein, the term "Cas protein" refers to an essential protein element in the CRISPR/Cas system, which is capable of forming a complex with two RNAs called CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). When it does, it forms an active endonuclease or nickase.

Cas gene and protein information can be obtained from GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto.

CRISPR-associated (cas) genes encoding Cas proteins are often associated with CRISPR repeat-spacer arrays. More than 40 different Cas protein families have been described. Among these protein families, Cas1 appears to be ubiquitous among the different CRISPR/Cas systems. There are three types of CRISPR-Cas systems. Among them, the type II CRISPR/Cas system involving Cas9 protein and crRNA and tracrRNA is representative and well known. Specific combinations of cas genes and repeat structures have been used to define eight CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube).

The Cas protein may be linked to a protein transduction domain. The protein transduction domain may be a poly-arginine domain or a TAT protein derived from HIV, but is not limited thereto.

The composition of the present invention may include a Cas element in the form of a protein or in the form of a nucleic acid encoding a Cas protein.

In the present invention, the Cas protein may be any Cas protein as long as it has endonuclease or nickase activity when forming a complex with the guide RNA.

Preferably, the Cas protein is a Cas9 protein or variant thereof.

A variant of the Cas9 protein may be a mutant form of Cas9 in which the catalytic aspartate residue is changed to any other amino acid. Preferably, the other amino acid may be, but is not limited to, alanine.

Further, the Cas9 protein may be, but is not limited to, a recombinant protein or isolated from an organism such as Streptococcus sp, preferably Streptococcus pyogens. Cas protein derived from Streptococcus pyogenes can recognize NGG trinucleotides.

As used herein, the term "guide RNA" refers to an RNA specific for a target DNA, capable of forming a complex with a Cas protein, and bringing the Cas protein to the target DNA.

In the present invention, the guide RNA may be composed of two RNAs, that is, CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or a single generated by fusion of essential parts of crRNA and tracrRNA. It may be single-chain RNA (sgRNA).

The guide RNA may be a dual RNA including crRNA and tracrRNA.

Any guide RNA may be used in the present invention as long as the guide RNA contains an essential part of crRNA and tracrRNA and a part complementary to the target.

The crRNA may hybridize with the target DNA.

The RGEN may consist of a Cas protein and a duplex RNA (constant tracrRNA and target-specific crRNA), or a Cas protein and sgRNA (fusion of an integral part of the constant tracrRNA and target-specific crRNA), replacing the crRNA It can be easily reprogrammed.

The guide RNA may further include one or more additional nucleotides at the 5' end of the crRNA of the single-stranded guide RNA or the double RNA.

Preferably, the guide RNA may further include two additional guanine nucleotides at the 5' end of the crRNA of the single-stranded guide RNA or the double RNA.

The guide RNA may be included in the composition in the form of RNA or DNA encoding the guide RNA. The guide RNA may be in the form of isolated RNA, RNA contained in a viral vector, or encoded in the vector. Preferably, the vector may be a viral vector, a plasmid vector, or an agrobacterium vector, but is not limited thereto.

As used herein, the term “cleavage” refers to the breakage of the covalent backbone of a nucleotide molecule.

In the present invention, the guide RNA may be prepared to be specific for any target to be cleaved. Accordingly, the composition of the present invention can cleave any target DNA by engineering or genotyping the target-specific portion of the guide RNA.

The guide RNA and Cas protein can act as a pair. As used herein, the term “paired Cas nickage” refers to a guide RNA and a Cas protein that function as a pair. A pair contains two guide RNAs. The guide RNA and Cas protein can act as a pair and induce two nicks in different DNA strands. The two nicks may be separated by at least 100 bps, but not limited thereto.

In the present invention, the composition can be used in vitro for detecting or diagnosing bacteria or viruses infected with cells.

In the present invention, the amplification method is not only a general PCR reaction, but also nested PCR, multiplex PCR, RT-PCR (reverse ranscriptase PCR), real time PCR, DOP (degenerate oligonucleotide primer) PCR, It can be used for quantitative RT-PCR, etc. In addition, it can be applied to all methods of amplifying a signal of a specific nucleotide sequence by primer selectivity and polymerization reaction of nucleic acid polymerase, so it can be used in various polymerase amplification tests such as rolling circle isothermal amplification and transcription isothermal amplification have.

As used herein, the term “sample” includes, but is not limited to, a sample for analysis such as tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine.

Claims (13)

generating a cleaved target nucleic acid fragment by reacting the target nucleic acid-specific guide RNA and the CAS protein with the target nucleic acid; and
A method of detecting a target nucleic acid comprising a; polymerase chain reaction (PCR) of the target nucleic acid fragment. The method of claim 1, wherein the guide RNA is designed so that the thermal denaturation temperature of the cleaved target nucleic acid is 90° C. or less. The method of claim 1, wherein the guide RNA is two or more guide RNA pairs. The method of claim 1, wherein the temperature of the thermal denaturation step in the polymerase chain reaction is higher than or equal to the thermal denaturation temperature of the target nucleic acid fragment and less than or equal to 92°C. The method of claim 1 , wherein the guide RNA is a single-stranded guide RNA. a guide RNA specific for a target nucleic acid or a DNA encoding the guide RNA; a Cas protein or a nucleic acid encoding the Cas protein; DNA polymerase; a pair of primers for amplification of a target nucleic acid; dNTPs; And a composition for detecting a nucleic acid comprising ^{Mg 2+.} The composition of claim 6, wherein the guide RNA is designed so that the thermal denaturation temperature of the cleaved target nucleic acid is 90°C or less. 7. The composition of claim 6, wherein the composition further comprises a fluorescent probe. 7. The composition of claim 6, wherein the composition further comprises a reverse transcriptase. 7. The composition of claim 6, wherein the composition further comprises a stabilizing agent. 11. The method of claim 10, wherein the stabilizing agent is selected from the group consisting of sorbitol, mannitol, galactitol, sucrose, trehalose and dextran. A composition characterized in that it is a carbohydrate. A kit for diagnosing a disease comprising the composition of any one of claims 6 to 11. The kit for diagnosing diseases according to claim 12, wherein the diagnostic kit is a cancer diagnostic kit, and the target nucleic acid includes a cancer-related gene mutation.