CN117431245A - CrRNA composition, obtaining method, product and application thereof - Google Patents
CrRNA composition, obtaining method, product and application thereof Download PDFInfo
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- CN117431245A CN117431245A CN202311315915.7A CN202311315915A CN117431245A CN 117431245 A CN117431245 A CN 117431245A CN 202311315915 A CN202311315915 A CN 202311315915A CN 117431245 A CN117431245 A CN 117431245A
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Abstract
The invention discloses a crRNA composition, a method for obtaining the crRNA composition and a product and application thereof, wherein the crRNA composition comprises the following components: at least three crrnas; wherein each of the crrnas targets the same target nucleic acid; any two recognition regions of the crRNA-targeted target nucleic acids do not overlap; a spacing region exists between the recognition regions of two adjacent crrnas, and the spacing regions of the target nucleic acids corresponding to the crRNA compositions are not less than two. The crRNA composition can accurately detect target nucleic acid in a sample, has high detection rate, low false negative rate and wide applicability, is particularly suitable for low-concentration target nucleic acid or degraded target nucleic acid, can effectively eliminate the influence of degradation on target nucleic acid detection, and has high application value.
Description
Technical Field
The invention relates to the field of nucleic acid detection, in particular to a crRNA composition, a method for obtaining the crRNA composition, a system, a composition, a kit and application thereof.
Background
In recent years, the nucleic acid detection method with high sensitivity and high specificity has increasingly obvious application value, and particularly has important application value in the fields of pathogen detection, early cancer screening, environmental microorganism detection and the like. Among them, quantitative reverse transcription polymerase chain reaction (RT-qPCR) has high detection sensitivity. In recent years, however, the advent of CRISPR-based nucleic acid detection techniques has promised to replace the traditional RT-qPCR method, with greater sensitivity. The technology mainly utilizes the side-cutting activity of CRISPR effect protein Cas, namely, after the Cas protein is combined with target RNA under the guidance of crRNA, the Cas protein can cut any single-stranded RNA molecule which is dissociated in the environment. At this time, a reporter molecule labeled with a fluorescent group and a quenching group is introduced into the system, and the activated Cas protein cleaves the reporter molecule and releases the fluorescent group, and the fluorescent detector detects the optical signal, thereby qualitatively or quantitatively detecting the target molecule.
However, current nucleic acid detection techniques for CRISPR, especially for degraded RNA molecules, remain to be investigated.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. To this end, the invention provides crRNA compositions, methods of obtaining crRNA compositions, systems, compositions, kits and uses thereof, and methods of detecting target nucleic acids. The crRNA composition can accurately detect target nucleic acid in a sample, has high detection rate, low false negative rate and wide applicability, is particularly suitable for low-concentration target nucleic acid or degraded target nucleic acid, can effectively eliminate the influence of degradation on target nucleic acid detection, and has high application value.
It is to be stated that the present invention has been completed based on the following work of the inventors:
for degraded nucleic acid samples (such as those containing the SARS-CoV-2N gene), the inventors have found that the effect of degradation on target nucleic acid detection cannot be eliminated, and the target nucleic acid detection rate is low, either by RT-qPCR detection methods or by using single or simple multiple crRNAs to detect target nucleic acids. The inventor discovers through a large number of creative experiments that when a specific crRNA composition is adopted to detect the degraded nucleic acid sample, the influence of degradation on the detection of the target nucleic acid can be effectively eliminated, and the initial target nucleic acid concentration of the sample is low and the detection rate is high. In particular, a particular crRNA composition needs to meet the following conditions: at least three crrnas are included, except that each crRNA targets the same target nucleic acid, the recognition regions of any two crrnas targeting the target nucleic acid do not overlap, and a spacer region exists between the recognition regions of two adjacent crrnas, and the spacer region of the target nucleic acid corresponding to the crRNA composition is not less than two. The inventors expect that optimizing the amount of crrnas in the crRNA composition not only effectively eliminates the effect of degradation on target nucleic acid detection, but also further improves the detection sensitivity. Therefore, the crRNA composition for detecting the target nucleic acid in the nucleic acid sample has the advantages of accurate result, high target nucleic acid detection rate, low false negative rate, high detection sensitivity and wide applicability, is particularly suitable for low-concentration target nucleic acid or degraded target nucleic acid, and can effectively eliminate the influence of degradation on target nucleic acid detection.
Thus, in a first aspect of the invention, the invention proposes a crRNA composition for targeting a target nucleic acid. The composition comprises: at least three crrnas; wherein each of the crrnas targets the same target nucleic acid; any two recognition regions of the crRNA-targeted target nucleic acids do not overlap; a spacing region exists between the recognition regions of two adjacent crrnas, and the spacing regions of the target nucleic acids corresponding to the crRNA compositions are not less than two. According to the embodiment of the invention, the crRNA composition can accurately detect the target nucleic acid in the sample, has high detection rate, low false negative rate and wide applicability, is particularly suitable for low-concentration target nucleic acid or degraded target nucleic acid, can effectively eliminate the influence of degradation on the detection of the target nucleic acid, and has high application value.
In a second aspect of the invention, the invention provides a method of obtaining a crRNA composition for targeting a target nucleic acid. The method comprises the following steps: providing sequence information of the target nucleic acid; designing a plurality of candidate crrnas targeting the target nucleic acid sequence based on sequence information of the target nucleic acid; screening at least three crrnas meeting a condition from the plurality of candidate crrnas as target crrnas: wherein the satisfaction condition includes: each of the target crrnas targets the same target nucleic acid; any two recognition regions of the target crRNA-targeting target nucleic acid do not overlap; a spacing region exists between the recognition regions of two adjacent target crrnas, and the spacing regions of all target crrnas corresponding to the target nucleic acids are not less than two. Thus, by the method of the invention described previously, a crRNA composition is obtained that targets a target nucleic acid. Furthermore, the crRNA composition can accurately detect target nucleic acid in a sample, has high detection rate, low false negative rate and wide applicability, is particularly suitable for low-concentration target nucleic acid or degraded target nucleic acid, can effectively eliminate the influence of degradation on target nucleic acid detection, and has high application value.
In a third aspect of the invention, the invention provides a system for detecting a target nucleic acid in a sample. The system comprises: the crRNA composition described above or a crRNA composition obtained according to the method described above. Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting target nucleic acids, are equally applicable to the system and are not described in detail herein.
In a fourth aspect of the invention, the invention provides a composition for detecting a target nucleic acid in a sample. The composition comprises: the crRNA composition described above or a crRNA composition obtained according to the method described above. Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting a target nucleic acid, are equally applicable to the compositions and are not described in detail herein.
In a fifth aspect of the invention, the invention provides a kit for detecting a target nucleic acid in a sample. The kit comprises: the aforementioned composition for detecting a target nucleic acid in a sample. Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting target nucleic acids, are equally applicable to the kit and are not described in detail herein.
In a sixth aspect of the invention, the invention provides the use of the crRNA composition described above, the system described above or the composition described above in the preparation of a kit. The kit is used for detecting target nucleic acid in a sample.
Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting target nucleic acids, are equally applicable to this application and will not be described in detail herein.
In a seventh aspect of the invention, the invention provides a method of detecting a target nucleic acid in a sample. The method comprises the following steps:
step 1: determining sequence information of the target nucleic acid;
step 2: a crRNA composition obtained according to the foregoing method or providing the foregoing crRNA composition;
step 3: reacting the crRNA composition, cas protein, and a reporter molecule for generating a detectable signal, collecting the detectable signal;
step 4: based on the detectable signal, the target nucleic acid is qualitatively or quantitatively analyzed.
Those skilled in the art will appreciate that the features and advantages described above for the crRNA composition, the method of obtaining a crRNA composition for targeting a target nucleic acid, are equally applicable to the method and are not described in detail herein.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a graph showing the results of RT-qPCR detection and Cas13 detection of a target RNA molecule of example 1 of the present invention with or without degradation, wherein (A) the fluorescence curve of RT-qPCR detection; (B) Quantitative results of fluorescence curves were detected using Cas13 of different numbers of crRNA molecules;
FIG. 2 is a graph showing the quantitative results of fluorescence curves of Cas13 detection using different crRNA combinations with or without degradation of the target RNA molecules of example 1 of the present invention;
FIG. 3 is a schematic diagram showing recognition regions and interval sequences of different crRNA targeting target nucleic acids in example 1 of the present invention, wherein 12, 3, 14, 2, 4 respectively represent recognition regions of five crRNAs 12, 3, 14, 2, and 4, respectively, the regions between the recognition regions of two adjacent crRNAs are interval regions, and the numbers above the interval regions represent the lengths of the interval regions;
FIG. 4 is a schematic diagram showing the recognition regions and spacer sequences of different crRNA targeting target nucleic acids in example 1 of the present invention, wherein 12, 3, 18, 13, 14, 2, 6, 4, 16, 17 represent the recognition regions of crRNA12, crRNA3, crRNA18, crRNA13, crRNA14, crRNA2, crRNA6, crRNA4, crRNA16, and crRNA17, respectively, the regions between the recognition regions of two adjacent crRNAs are spacer regions, and the numbers above the spacer regions represent the lengths of the spacer regions;
FIG. 5 is a graph showing the results of RT-qPCR detection and Cas13 detection of the target RNA molecule of example 2 of the present invention under the condition of degradation or non-degradation, respectively, wherein (A) the fluorescence curve of RT-qPCR detection is that of N.T group is a negative control group, and water is used as a substrate for detection; (B) Fluorescence curve quantification of Cas13 detection using 5 crRNA molecules, the 5 crrnas used were crRNA12, crRNA3, crRNA14, crRNA2, crRNA4, respectively.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
Terms and definitions
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
Herein, the term "CRISPR" refers to clustered, regularly interspaced short palindromic repeats (Clustered regularly interspaced short palindromic repeats) from the immune system of a microorganism.
In this context, the term "crRNA" is equivalent to "CRISPR-extended RNA", "CRISPRRNA". The Type VI family gene locus contains a CRISPR array consisting of a Repeat sequence (Repeat) and a spacer sequence (spacer), which transcribes to form an immature pre-crRNA, which is cleaved to form a mature crRNA, leading to the Cas protein functioning.
As used herein, the term "target nucleic acid" refers to a polynucleotide molecule extracted from a biological sample (sample to be tested).
The terms "biological sample" and "sample to be tested" are collectively referred to herein as a sample. For example, the sample may be any solid or fluid sample obtained, excreted or secreted from any organism, including but not limited to single cell organisms such as bacteria, yeasts, protozoa, amoebas and the like, multicellular organisms (e.g. plants or animals, including samples from healthy or surface healthy human subjects or human patients affected by the condition or disease to be diagnosed or investigated, e.g. infection by pathogenic microorganisms such as pathogenic bacteria or viruses). For example, the sample may also be a biological fluid obtained from, for example, blood, plasma, serum, urine, stool, sputum, mucus, lymph, synovial fluid, bile, ascites, pleural effusion, seroma, saliva, cerebrospinal fluid, aqueous or vitreous humor, or any bodily secretion, exudate (e.g., fluid obtained from an abscess or any other site of infection or inflammation) or a fluid obtained from a joint (e.g., a normal joint or a joint affected by a disease, such as rheumatoid arthritis, osteoarthritis, gout, or septic arthritis), or a swab of a skin or mucosal surface. The sample may also be a sample obtained from any organ or tissue (including a biopsy or autopsy specimen, such as a tumor biopsy) or may comprise cells (primary cells or cultured cells) or a medium conditioned by any cell, tissue or organ. Exemplary samples include, but are not limited to, cells, cell lysates, blood smears, cell centrifuge preparations, cytological smears, bodily fluids (e.g., blood, plasma, serum, saliva, sputum, urine, bronchoalveolar lavage, semen, etc.), tissue biopsies (e.g., tumor biopsies), fine needle aspirates, and/or tissue sections (e.g., cryostat tissue sections and/or paraffin embedded tissue sections).
Herein, the term "Cas protein" refers to a CRISPR associated protein, which upon binding to the feature sequence (target sequence) to be detected (i.e. forming a ternary complex of Cas protein gRNA target sequence), can induce its parachuting activity, i.e. randomly cleaving non-targeted single stranded nucleotides (i.e. the reporter molecules described herein, sometimes also referred to as "single stranded nucleic acid detectors"). When the Cas protein binds to a signature sequence, it may induce its bypass activity, either with or without cleavage of the signature sequence. It induces its parachuting activity, usually by cleaving a characteristic sequence. More specifically, it induces its parachuting activity by cleaving the single stranded signature sequence. In some cases, the Cas protein recognizes the signature sequence by recognizing PAM (protospacer adjacent motif) adjacent to the signature sequence.
The invention provides a crRNA composition for targeting a target nucleic acid, a method for obtaining the same, a system for detecting a target nucleic acid in a nucleic acid sample, a composition, a kit and applications thereof, which are described in detail below.
CrRNA compositions
The present invention provides a crRNA composition for targeting a target nucleic acid. The composition comprises: at least three crrnas; wherein each of the crrnas targets the same target nucleic acid; any two recognition regions of the crRNA-targeted target nucleic acids do not overlap; a spacing region exists between the recognition regions of two adjacent crrnas, and the spacing regions of the target nucleic acids corresponding to the crRNA compositions are not less than two. According to the embodiment of the invention, the crRNA composition can accurately detect the target nucleic acid in the sample, has high detection rate, low false negative rate and wide applicability, is particularly suitable for low-concentration target nucleic acid or degraded target nucleic acid, can effectively eliminate the influence of degradation on the detection of the target nucleic acid, and has high application value.
As used herein, a "recognition region of a crRNA" is equivalent to a "target sequence of a crRNA" and refers to a target sequence of a crRNA targeted target nucleic acid. In this context, the spacer sequence of the corresponding crRNA. The crRNA comprises two parts, namely a DR (Direct Repeat, DR) and a Spacer (Spacer), wherein the DR of the crRNA is a scaffold to which the Cas13 protein binds, and the Spacer of the crRNA guides the Cas13 protein to its target sequence in a base complementary manner and binds complementarily to the target sequence.
In this context, a "spacer region" refers to a nucleotide sequence that exists between the recognition regions of two adjacent crrnas of a target nucleic acid.
According to an embodiment of the invention, the recognition region of the crRNA is 17 to 28 nucleotides in length. Preferably, the recognition region of the crRNA is 19 to 28 nucleotides in length. Thus, the crRNA composition can effectively recognize target nucleic acids and their degradants in a nucleic acid sample. Illustratively, the recognition region of the crRNA is 20, 21, 22, 23, 24, 25, 26, 27 nucleotides in length.
According to an embodiment of the invention, the length of the spacer region is not less than 30 nucleotides. Preferably, the spacer region is 50 to 250 nucleotides in length, more preferably 100 to 200 nucleotides in length. According to embodiments of the invention, the length of the spacer region is important for the crRNA composition to efficiently recognize target nucleic acids and their degradants in a nucleic acid sample. The inventor finds through a large number of experiments that when the length of a spacing region of the crRNA composition is proper, ternary complexes formed by Cas proteins, crRNAs and nucleic acid substrates which target different positions are not easy to compete with each other in space, and the Cas proteins are beneficial to exerting nucleic acid cleavage activity. For example, if the length of the spacer region is too short, the adjacent two recognition regions are very small apart, and steric hindrance exists between the ternary complexes, thereby affecting the Cas protein to exert nucleic acid cleavage activity. When the length of the interval region is not less than 30 nucleotides, the detection effect is better, the detection rate is high, and the method is particularly suitable for detecting low-concentration target nucleic acid and degraded target nucleic acid.
According to an embodiment of the invention, the sum of the lengths of the recognition and spacer regions corresponding to the crRNA composition is 30-100% of the total length of the target nucleic acid. Thus, the crRNA composition is used for detecting target nucleic acid in a nucleic acid sample, and the initial target nucleic acid concentration of the sample is low and the detection rate is high.
According to an embodiment of the invention, the sum of the lengths of the recognition and spacer regions corresponding to the crRNA composition is 50-100% of the total length of the target nucleic acid. Thus, the crRNA composition is used for detecting target nucleic acid in a nucleic acid sample, and the initial target nucleic acid concentration of the sample is low and the detection rate is high.
According to an embodiment of the invention, the sum of the lengths of the recognition and spacer regions corresponding to the crRNA composition is 60-100% of the total length of the target nucleic acid. Thus, the crRNA composition is used for detecting target nucleic acid in a nucleic acid sample, and the initial target nucleic acid concentration of the sample is low and the detection rate is high. According to the embodiment of the invention, when the sum of the lengths of the recognition region and the interval region corresponding to the crRNA composition is within the range, not only the influence of degradation on the detection of target nucleic acid can be effectively eliminated, but also the detection sensitivity of the CRISPR-based nucleic acid detection technology can be improved. Therefore, the crRNA composition can be used for detecting target nucleic acid in a degraded nucleic acid sample, and has the advantages of accurate result, high target nucleic acid detection rate, low false negative misjudgment rate, high detection sensitivity and high clinical application value.
In some embodiments of the invention, the target nucleic acid targeted by the crRNA composition may be one or more target molecules as a diagnostic of a disease. In some specific embodiments, the disease may be an infection, an organ disease, a blood disease, an immune system disease, a cancer, a brain and nervous system disease, an endocrine disease, a pregnancy or labor related disease, a genetic disease, or an environmental acquired disease. In other specific embodiments, the disease may be cancer or an autoimmune disease or infection.
In some embodiments of the invention, the target nucleic acid targeted by the crRNA composition may be at least one target molecule comprising a cancer specific somatic mutation. Cancer-specific mutations may confer resistance. The drug resistance mutation may be induced by treatment with ibrutinib (ibrutinib), erlotinib (erlotinib), imatinib (imatinib), gefitinib (gefitinib), crizotinib (crizotinib), trastuzumab (trastuzumab), vemurafenib (vemurafenib), RAF/MEK, checkpoint blocking therapy or antiestrogenic therapy. The cancer-specific mutations may be present in one or more genes encoding a protein selected from the group consisting of: programmed death ligand 1 (PD-L1), androgen Receptor (AR), bruton's Tyrosine Kinase (BTK), epidermal Growth Factor Receptor (EGFR), BCR-Abl, c-kit, PIK3CA, HER2, EML4-ALK, KRAS, ALK, ROS1, AKT1, BRAF, MEK1, MEK2, NRAS, RAC1 and ESR1. The cancer specific mutation may be a mutation in a gene selected from the group consisting of: CASP8, B2M, PIK3CA, SMC1A, ARID5B, TET, ALPK2, COL5A1, TP53, dnar, NCOR1, MORC4, CIC, IRF6, MYOCD, ANKLE1, CNKSR1, NF1, SOS1, ARID2, CUL4B, DDX3X, FUBP1, TCP11L2, HLA-A, B or C, CSNK A1, MET, ASXL1, PD-L2, IDO1, IDO2, ALOX12B and ALOX15B, or copy number increase, excluding whole chromosome events affecting any of the following chromosome bands: 6q16.1-q21, 6q22.31-q24.1, 6q25.1-q26, 7p11.2-q11.1, 8p23.1, 8p11.23-p11.21 (containing IDO1, IDO 2), 9p24.2-p23 (containing PDL1, PDL 2), 10p15.3, 10p15.1-p13, 11p14.1, 12p13.32-p13.2, 17p13.1 (containing ALOX12B, ALOX B) and 22q11.1-q11.21.
In some embodiments of the invention, the target nucleic acid targeted by the crRNA composition may be at least one target molecule comprising a loss of heterozygosity (LOH) marker.
In some embodiments of the invention, the target nucleic acid targeted by the crRNA composition may be at least one target molecule comprising a Single Nucleotide Polymorphism (SNP).
In some embodiments of the invention, the infection is caused by a virus, bacteria or fungus.
In some embodiments of the invention, the infection is a viral infection. In particular embodiments, the viral infection is caused by a double stranded RNA virus, a sense RNA virus, an antisense RNA virus, a retrovirus, or a combination thereof, or the viral infection is caused by: coronaviridae (Coronaviridae) viruses, picornaviridae (Picornaviridae) viruses, calicividae (Caliciviridae) viruses, flaviviridae (Flaviviridae) viruses, togaviridae (Togaviridae) viruses, bornaviridae (Bornaviridae), filoviridae (Filoviridae), paramyxoviridae (Paramyxoviridae), alveolar viridae (Pneumoviridae), rhabdoviridae (Rhabdoviridae), arenaviridae (Arenaviridae), bunyaviridae (Bunyaviridae), orthomyxoviridae (Orthomexoviridae) or butyl type viruses (Deltaviruses), or viral infections are caused by: coronavirus, SARS, polio virus, rhinovirus, hepatitis A, norwalk virus, yellow fever virus, west Nile virus, hepatitis C virus, dengue virus (Denguefevervirus), zikavirus, rubella virus, ross river virus (Rossrriverus), sindbis virus (Sindbis virus), chikungunya virus (Chikungunyavirus), borna virus (Bernarvieae virus), ebola virus (Ebola virus), marburg virus (Marburg virus), measles virus, mumps virus, nipa virus (Nipah virus), hendee virus (Hendrus virus), newcastle disease virus, conn respiratory syncytial virus, rabies virus, lassa virus (Lassav virus), hantavirus (Hantaus), crinary virus (Crinary virus), or hepatitis virus-virus.
According to an embodiment of the invention, the target nucleic acid is RNA, preferably RNA derived from an RNA virus.
It should be noted that the nucleotide sequences described in the present invention are all shown in a 5 '-end to 3' -end manner.
According to an embodiment of the invention, the target nucleic acid is the N gene of SARS-COV-2 virus. Preferably, the target nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 1.
AUGUCUGAUAAUGGACCCCAAAAUCAGCGAAAUGCACCCCGCAUUACGUUUGGUGGACCCUCAGAUUCAACUGGCAGUAACCAGAAUGGAGAACGCAGUGGGGCGCGAUCAAAACAACGUCGGCCCCAAGGUUUACCCAAUAAUACUGCGUCUUGGUUCACCGCUCUCACUCAACAUGGCAAGGAAGACCUUAAAUUCCCUCGAGGACAAGGCGUUCCAAUUAACACCAAUAGCAGUCCAGAUGACCAAAUUGGCUACUACCGAAGAGCUACCAGACGAAUUCGUGGUGGUGACGGUAAAAUGAAAGAUCUCAGUCCAAGAUGGUAUUUCUACUACCUAGGAACUGGGCCAGAAGCUGGACUUCCCUAUGGUGCUAACAAAGACGGCAUCAUAUGGGUUGCAACUGAGGGAGCCUUGAAUACACCAAAAGAUCACAUUGGCACCCGCAAUCCUGCUAACAAUGCUGCAAUCGUGCUACAACUUCCUCAAGGAACAACAUUGCCAAAAGGCUUCUACGCAGAAGGGAGCAGAGGCGGCAGUCAAGCCUCUUCUCGUUCCUCAUCACGUAGUCGCAACAGUUCAAGAAAUUCAACUCCAGGCAGCAGUAGGGGAACUUCUCCUGCUAGAAUGGCUGGCAAUGGCGGUGAUGCUGCUCUUGCUUUGCUGCUGCUUGACAGAUUGAACCAGCUUGAGAGCAAAAUGUCUGGUAAAGGCCAACAACAACAAGGCCAAACUGUCACUAAGAAAUCUGCUGCUGAGGCUUCUAAGAAGCCUCGGCAAAAACGUACUGCCACUAAAGCAUACAAUGUAACACAAGCUUUCGGCAGACGUGGUCCAGAACAAACCCAAGGAAAUUUUGGGGACCAGGAACUAAUCAGACAAGGAACUGAUUACAAACAUUGGCCGCAAAUUGCACAAUUUGCCCCCAGCGCUUCAGCGUUCUUCGGAAUGUCGCGCAUUGGCAUGGAAGUCACACCUUCGGGAACGUGGUUGACCUACACAGGUGCCAUCAAAUUGGAUGACAAAGAUCCAAAUUUCAAAGAUCAAGUCAUUUUGCUGAAUAAGCAUAUUGACGCAUACAAAACAUUCCCACCAACAGAGCCUAAAAAGGACAAAAAGAAGAAGGCUGAUGAAACUCAAGCCUUACCGCAGAGACAGAAGAAACAGCAAACUGUGACUCUUCUUCCUGCUGCAGAUUUGGAUGAUUUCUCCAAACAAUUGCAACAAUCCAUGAGCAGUGCUGACUCAACUCAGGCCUAA(SEQ ID NO:1)。
According to an embodiment of the invention, the crRNA composition comprises at least three crRNAs and has at least three of the nucleotide sequences as shown in SEQ ID NOs 2 to 11. Preferably, the crRNA composition is three crRNAs and has a nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 8.
CUCUCAAGCUGGUUCAAUCUGUCA(SEQ ID NO:2)。
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
UGUAGCACGAUUGCAGCAUU(SEQ ID NO:8)。
According to an embodiment of the invention, the crRNA composition comprises at least four crRNAs and has at least four of the nucleotide sequences as shown in SEQ ID NOs 2 to 11. Preferably, the crRNA composition is four crRNAs and has a nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8.
CUCUCAAGCUGGUUCAAUCUGUCA(SEQ ID NO:2)。
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
UCUGGUUACUGCCAGUUGAA(SEQ ID NO:6)。
UGUAGCACGAUUGCAGCAUU(SEQ ID NO:8)。
According to an embodiment of the invention, the crRNA composition comprises at least five crRNAs and has at least five of the nucleotide sequences as shown in SEQ ID NOs 2 to 11.
Preferably, the crRNA composition is five crRNAs and has nucleotide sequences as shown in SEQ ID NO. 2-4, SEQ ID NO. 6, and SEQ ID NO. 8. CUCUCAAGCUGGUUCAAUCUGUCA (SEQ ID NO: 2).
GUCAUCUGGACUGCUAUUGG(SEQ ID NO:3)。
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
UCUGGUUACUGCCAGUUGAA(SEQ ID NO:6)。
UGUAGCACGAUUGCAGCAUU(SEQ ID NO:8)。
Preferably, the crRNA composition is five crRNAs and has nucleotide sequences as shown in SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 9-11.
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
UCUGGUUACUGCCAGUUGAA(SEQ ID NO:6)。
GAAGCGCUGGGGGCAAAUUG(SEQ ID NO:9)。
AUGCGCGACAUUCCGAAGAA(SEQ ID NO:10)。
UUGGUGUAUUCAAGGCUCCC(SEQ ID NO:11)。
According to an embodiment of the invention, the crRNA composition is ten crRNAs and has the nucleotide sequence as shown in SEQ ID NOs 2 to 11.
CUCUCAAGCUGGUUCAAUCUGUCA(SEQ ID NO:2)。
GUCAUCUGGACUGCUAUUGG(SEQ ID NO:3)。
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
CCAGACAUUUUGCUCUCAAG(SEQ ID NO:5)。
UCUGGUUACUGCCAGUUGAA(SEQ ID NO:6)。
GGAUUGCGGGUGCCAAUGUG(SEQ ID NO:7)。
UGUAGCACGAUUGCAGCAUU(SEQ ID NO:8)。
GAAGCGCUGGGGGCAAAUUG(SEQ ID NO:9)。
AUGCGCGACAUUCCGAAGAA(SEQ ID NO:10)。
UUGGUGUAUUCAAGGCUCCC(SEQ ID NO:11)。
Method
The present invention provides a method of obtaining a crRNA composition for targeting a target nucleic acid. The method comprises the following steps: providing sequence information of the target nucleic acid; designing a plurality of candidate crrnas targeting the target nucleic acid sequence based on sequence information of the target nucleic acid; screening at least three crrnas meeting a condition from the plurality of candidate crrnas as target crrnas: wherein the satisfaction condition includes: each of the target crrnas targets the same target nucleic acid; any two recognition regions of the target crRNA-targeting target nucleic acid do not overlap; a spacing region exists between the recognition regions of two adjacent target crrnas, and the spacing regions of all target crrnas corresponding to the target nucleic acids are not less than two. Thus, by the method of the invention described previously, a crRNA composition is obtained that targets a target nucleic acid. Furthermore, the crRNA composition can accurately detect target nucleic acid in a sample, has high detection rate, low false negative rate and wide applicability, is particularly suitable for low-concentration target nucleic acid or degraded target nucleic acid, can effectively eliminate the influence of degradation on target nucleic acid detection, and has high application value.
According to an embodiment of the invention, the recognition region of the target crRNA is 17 to 28 nucleotides in length. Preferably, the recognition region of the crRNA is 19 to 28 nucleotides in length. Thus, the crRNA composition of the embodiment of the invention can be further obtained, which can effectively recognize the target nucleic acid and the degradation product thereof in the nucleic acid sample. Illustratively, the recognition region of the crRNA is 20, 21, 22, 23, 24, 25, 26, 27 nucleotides in length.
According to an embodiment of the invention, the length of the spacer region is not less than 30 nucleotides. Preferably, the spacer region is 50 to 250 nucleotides in length, more preferably 100 to 200 nucleotides in length. Thus, the crRNA composition of the embodiment of the invention can be further obtained, which can effectively recognize the target nucleic acid and the degradation product thereof in the nucleic acid sample. Illustratively, the spacer region is 60, 70, 80, 90, 100, 110, 120, 130, 14, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 nucleotides in length.
According to an embodiment of the invention, the sum of the lengths of the recognition region and the interval region corresponding to all target crrnas is 30-100% of the total length of the target RNA sequence. Thus, the crRNA composition of the embodiment of the invention can be further obtained, which can effectively recognize the target nucleic acid and the degradation product thereof in the nucleic acid sample. Illustratively, the sum of the lengths of the recognition and spacer regions corresponding to all target crrnas is 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the total length of the target RNA sequence.
According to an embodiment of the invention, the sum of the lengths of the recognition region and the interval region corresponding to all target crrnas is 50-100% of the total length of the target RNA sequence. Thus, the crRNA composition of the embodiment of the invention can be further obtained, which can effectively recognize the target nucleic acid and the degradation product thereof in the nucleic acid sample. Illustratively, the sum of the lengths of the recognition and spacer regions corresponding to all target crrnas is 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% of the total length of the target RNA sequence.
According to an embodiment of the invention, the sum of the lengths of the recognition region and the interval region corresponding to all target crrnas is 60-100% of the total length of the target RNA sequence. Thus, the crRNA composition of the embodiment of the invention can be further obtained, which can effectively recognize the target nucleic acid and the degradation product thereof in the nucleic acid sample. Illustratively, the sum of the lengths of the recognition and spacer regions corresponding to all target crrnas is 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% of the total length of the target RNA sequence.
In some embodiments of the invention, the target crRNA may be designed as a target molecule for disease diagnosis. In some specific embodiments, the disease may be an infection, an organ disease, a blood disease, an immune system disease, a cancer, a brain and nervous system disease, an endocrine disease, a pregnancy or labor related disease, a genetic disease, or an environmental acquired disease. In other specific embodiments, the disease may be cancer or an autoimmune disease or infection.
It should be noted that the features and advantages described above for the crRNA composition for targeting a target nucleic acid are equally applicable to the method of obtaining the crRNA composition for targeting a target nucleic acid, and are not described in detail herein.
The target nucleic acid is RNA. RNA derived from RNA viruses is preferred.
According to an embodiment of the invention, the target nucleic acid is the N gene of SARS-COV-2 virus. Preferably, the target nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 1.
AUGUCUGAUAAUGGACCCCAAAAUCAGCGAAAUGCACCCCGCAUUACGUUUGGUGGACCCUCAGAUUCAACUGGCAGUAACCAGAAUGGAGAACGCAGUGGGGCGCGAUCAAAACAACGUCGGCCCCAAGGUUUACCCAAUAAUACUGCGUCUUGGUUCACCGCUCUCACUCAACAUGGCAAGGAAGACCUUAAAUUCCCUCGAGGACAAGGCGUUCCAAUUAACACCAAUAGCAGUCCAGAUGACCAAAUUGGCUACUACCGAAGAGCUACCAGACGAAUUCGUGGUGGUGACGGUAAAAUGAAAGAUCUCAGUCCAAGAUGGUAUUUCUACUACCUAGGAACUGGGCCAGAAGCUGGACUUCCCUAUGGUGCUAACAAAGACGGCAUCAUAUGGGUUGCAACUGAGGGAGCCUUGAAUACACCAAAAGAUCACAUUGGCACCCGCAAUCCUGCUAACAAUGCUGCAAUCGUGCUACAACUUCCUCAAGGAACAACAUUGCCAAAAGGCUUCUACGCAGAAGGGAGCAGAGGCGGCAGUCAAGCCUCUUCUCGUUCCUCAUCACGUAGUCGCAACAGUUCAAGAAAUUCAACUCCAGGCAGCAGUAGGGGAACUUCUCCUGCUAGAAUGGCUGGCAAUGGCGGUGAUGCUGCUCUUGCUUUGCUGCUGCUUGACAGAUUGAACCAGCUUGAGAGCAAAAUGUCUGGUAAAGGCCAACAACAACAAGGCCAAACUGUCACUAAGAAAUCUGCUGCUGAGGCUUCUAAGAAGCCUCGGCAAAAACGUACUGCCACUAAAGCAUACAAUGUAACACAAGCUUUCGGCAGACGUGGUCCAGAACAAACCCAAGGAAAUUUUGGGGACCAGGAACUAAUCAGACAAGGAACUGAUUACAAACAUUGGCCGCAAAUUGCACAAUUUGCCCCCAGCGCUUCAGCGUUCUUCGGAAUGUCGCGCAUUGGCAUGGAAGUCACACCUUCGGGAACGUGGUUGACCUACACAGGUGCCAUCAAAUUGGAUGACAAAGAUCCAAAUUUCAAAGAUCAAGUCAUUUUGCUGAAUAAGCAUAUUGACGCAUACAAAACAUUCCCACCAACAGAGCCUAAAAAGGACAAAAAGAAGAAGGCUGAUGAAACUCAAGCCUUACCGCAGAGACAGAAGAAACAGCAAACUGUGACUCUUCUUCCUGCUGCAGAUUUGGAUGAUUUCUCCAAACAAUUGCAACAAUCCAUGAGCAGUGCUGACUCAACUCAGGCCUAA(SEQ ID NO:1)。
According to an embodiment of the invention, the crRNA composition obtained comprises at least three crRNAs and has at least three of the nucleotide sequences as shown in SEQ ID NOs 2 to 11. Preferably, the crRNA composition obtained is three crRNAs and has the nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 8.
CUCUCAAGCUGGUUCAAUCUGUCA(SEQ ID NO:2)。
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
UGUAGCACGAUUGCAGCAUU(SEQ ID NO:8)。
According to an embodiment of the invention, the crRNA composition obtained comprises at least four crRNAs and has at least four of the nucleotide sequences as shown in SEQ ID NOs 2 to 11. Preferably, the crRNA composition obtained is four crRNAs and has a nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8.
CUCUCAAGCUGGUUCAAUCUGUCA(SEQ ID NO:2)。
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
UCUGGUUACUGCCAGUUGAA(SEQ ID NO:6)。
UGUAGCACGAUUGCAGCAUU(SEQ ID NO:8)。
According to an embodiment of the invention, the crRNA composition obtained comprises at least five crRNAs and has at least five of the nucleotide sequences as shown in SEQ ID NOs 2 to 11.
Preferably, the crRNA composition obtained is five crRNAs and has nucleotide sequences as shown in SEQ ID NO. 2-4, SEQ ID NO. 6, SEQ ID NO. 8.
CUCUCAAGCUGGUUCAAUCUGUCA(SEQ ID NO:2)。
GUCAUCUGGACUGCUAUUGG(SEQ ID NO:3)。
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
UCUGGUUACUGCCAGUUGAA(SEQ ID NO:6)。
UGUAGCACGAUUGCAGCAUU(SEQ ID NO:8)。
Preferably, the crRNA composition obtained is five crRNAs and has nucleotide sequences as shown in SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 9-11.
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
UCUGGUUACUGCCAGUUGAA(SEQ ID NO:6)。
GAAGCGCUGGGGGCAAAUUG(SEQ ID NO:9)。
AUGCGCGACAUUCCGAAGAA(SEQ ID NO:10)。
UUGGUGUAUUCAAGGCUCCC(SEQ ID NO:11)。
According to an embodiment of the present invention, the target crRNA composition obtained is ten crRNAs and has the nucleotide sequences as shown in SEQ ID NOs 2 to 11.
CUCUCAAGCUGGUUCAAUCUGUCA(SEQ ID NO:2)。
GUCAUCUGGACUGCUAUUGG(SEQ ID NO:3)。
UUUGCGGCCAAUGUUUGUAA(SEQ ID NO:4)。
CCAGACAUUUUGCUCUCAAG(SEQ ID NO:5)。
UCUGGUUACUGCCAGUUGAA(SEQ ID NO:6)。
GGAUUGCGGGUGCCAAUGUG(SEQ ID NO:7)。
UGUAGCACGAUUGCAGCAUU(SEQ ID NO:8)。
GAAGCGCUGGGGGCAAAUUG(SEQ ID NO:9)。
AUGCGCGACAUUCCGAAGAA(SEQ ID NO:10)。
UUGGUGUAUUCAAGGCUCCC(SEQ ID NO:11)。
According to an embodiment of the invention, the designing of the plurality of candidate crrnas targeting the target nucleic acid sequence is performed using crRNA design software.
As known to those skilled in the art, the design of crrnas is one of the key steps in CRISPR nucleic acid detection technology. To obtain candidate crrnas, one skilled in the art can resort to crRNA sequence design tools such as various commonly used crRNA sequence design and optimization software. In software predictions, it is a routine strategy in the art to screen for predicted crrnas with higher scores in order to obtain the desired crrnas.
According to an embodiment of the invention, the crRNA design software is Cas13design.
System and method for controlling a system
The present invention provides a system for detecting a target nucleic acid in a sample. The system comprises: the crRNA composition described above or a crRNA composition obtained according to the method described above. Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting target nucleic acids, are equally applicable to the system and are not described in detail herein.
In some embodiments of the invention, the system further comprises: cas proteins and reporter molecules.
In some embodiments of the invention, the Cas protein is a Cas13a protein.
In some embodiments of the invention, the Cas13a protein is selected from one of LbuCas13a, lwcas 13a, lbcas 13a, camCas13a, lshCas13a, rcaCas13a, hhcas 13a, pprCas13a, lseCas13a, lbmCas13a, or lbnncas 13 a.
In some embodiments of the invention, the Cas protein is a Cas12a protein.
In some embodiments of the invention, the Cas12a protein is selected from one of AsCas12a, lbCas12a, lb4Cas12a, lb5Cas12a, fnCas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, or BoCas12 a.
In some embodiments of the invention, the reporter molecule is a DNA or RNA single stranded nucleic acid detector. The single-stranded nucleic acid detector comprises a reporter group or a marker molecule that does not exhibit a reporter signal when in an initial state (i.e., not cleaved), and exhibits a detectable signal when the single-stranded nucleic acid detector is cleaved, i.e., exhibits a detectable distinction between after cleavage and before cleavage. Reflecting that the target nucleic acid contains a target sequence to be detected if a detectable difference can be detected; alternatively, if the detectable difference cannot be detected, it is reflected that the target nucleic acid does not contain the target sequence to be detected. Herein, if a detectable difference can be detected, it is reflected in the detection of the inclusion of the target nucleic acid in the sample; alternatively, if the detectable difference cannot be detected, it is reflected that the target nucleic acid is not contained in the test sample.
In some embodiments of the invention, the reporter group or marker molecule, illustratively, includes, but is not limited to: a fluorescent group and a quenching group, wherein the fluorescent group is selected from one or any several of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, texas Red or LCRED 460; the quenching group is selected from one or more of BHQ1, BHQ2, BHQ3, dabcy1 or Tamra.
Composition and method for producing the same
The present invention provides a composition for detecting a target nucleic acid in a sample. The composition comprises: the crRNA composition described above or a crRNA composition obtained according to the method described above.
In some embodiments of the invention, the method further comprises: cas proteins and reporter molecules.
In some embodiments of the invention, the Cas protein is a Cas13a protein.
In some embodiments of the invention, the Cas13a protein is selected from one of LbuCas13a, lwcas 13a, lbcas 13a, camCas13a, lshCas13a, rcaCas13a, hhcas 13a, pprCas13a, lseCas13a, lbmCas13a, or lbnncas 13 a.
In some embodiments of the invention, the Cas protein is a Cas12a protein.
In some embodiments of the invention, the Cas12a protein is selected from one of AsCas12a, lbCas12a, lb4Cas12a, lb5Cas12a, fnCas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, or BoCas12 a.
Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting a target nucleic acid, are equally applicable to the above-described compositions and are not described in detail herein.
Kit for detecting a substance in a sample
The present invention provides a kit for detecting a target nucleic acid in a sample. The kit comprises: the aforementioned composition for detecting a target nucleic acid in a sample. In accordance with an embodiment of the present invention,
in some embodiments of the invention, the kit further comprises: cas proteins and reporter molecules.
In some embodiments of the invention, the Cas protein is a Cas13a protein.
In some embodiments of the invention, the Cas13a protein is selected from one of LbuCas13a, lwcas 13a, lbcas 13a, camCas13a, lshCas13a, rcaCas13a, hhcas 13a, pprCas13a, lseCas13a, lbmCas13a, or lbnncas 13 a.
In some embodiments of the invention, the Cas protein is a Cas12a protein.
In some embodiments of the invention, the Cas12a protein is selected from one of AsCas12a, lbCas12a, lb4Cas12a, lb5Cas12a, fnCas12a, hkCas12a, osCas12a, tsCas12a, bbCas12a, or BoCas12 a.
Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting target nucleic acids, are equally applicable to the above-described kits and are not described in detail herein.
Application of
The invention provides the use of the crRNA composition described above, the system described above or the composition described above in the preparation of a kit for detecting a target nucleic acid in a sample.
According to an embodiment of the invention, the target nucleic acid is derived from a microorganism, soil, water source, animal or plant sample.
According to an embodiment of the invention, the target nucleic acid is derived from a virus or a bacterium.
According to an embodiment of the invention, the target nucleic acid comprises at least one of RNA, ASO and a nucleic acid aptamer.
According to an embodiment of the invention, the target nucleic acid comprises at least one of mRNA, miRNA, and siRNA.
According to an embodiment of the invention, the target nucleic acid is derived from RNA of an RNA virus.
According to an embodiment of the invention, the target nucleic acid is the N gene of SARS-COV-2 virus.
Preferably, the target nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 1.
Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting target nucleic acids, are equally applicable to the above-described applications and are not described in detail herein.
Method
The present invention provides a method for detecting a target nucleic acid in a sample. The method comprises the following steps: step 1: determining sequence information of the target nucleic acid;
Step 2: a crRNA composition obtained according to the foregoing method or providing the foregoing crRNA composition;
step 3: reacting the crRNA composition, cas protein, and a reporter molecule for generating a detectable signal, collecting the detectable signal;
step 4: based on the detectable signal, the target nucleic acid is qualitatively or quantitatively analyzed.
According to an embodiment of the invention, step 3 of the method comprises: a reaction system is constructed based on the system, the composition or the kit, and a reaction is performed to collect a detectable signal.
Those skilled in the art will appreciate that the features and advantages described above for crRNA compositions, methods of obtaining crRNA compositions for targeting target nucleic acids, are equally applicable to the above methods and are not described in detail herein.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the examples of the present invention, for ease of investigation, a degraded sample of a target RNA molecule is obtained by the following method, unless specifically stated:
(1) Adding the target RNA molecule to 1X Isothermal Amplification Buffer, and heating at 95 ℃ for 1 hour; or adding the target RNA molecule into 0.1M NaOH solution, and heating at 95 ℃ for 60-150 min.
(2) For the above-described degradation-treated RNA molecules, RT-qPCR was used to quantify the target RNA molecules to assess the extent of degradation.
If not specified, RT-qPCR can detect target RNA molecules, but the number of RT-qPCR cycles is significantly increased in the degradation samples, in the present embodiment described as "degraded samples"; RT-qPCR cannot detect degraded samples of target RNA molecules, and is described as "severely degraded samples" in the examples of the present invention.
Example 1: detection of degraded SARS-CoV-2N Gene Using the method of the present invention
In this example, NCBI was used to obtain SARS-CoV-2N gene sequence information and the degraded SARS-CoV-2N gene was detected as follows.
In this example, the degraded SARS-CoV-2N gene (target RNA molecule) is obtained by the following method: the target RNA molecules were added to 1X Isothermal Amplification Buffer and heated at 95℃for 1 hour. The degraded SARS-CoV-2N gene adopts RT-qPCR to quantify target RNA molecule, and can detect target RNA molecule, but the number of RT-qPCR cycle is obviously increased.
(1) Design of crRNA molecules targeting SARS-CoV-2N Gene
Cas13design is adopted for SARS-CoV-2N target sequence 1 The software designed multiple crRNA molecules targeting the SARS-CoV-2N gene.
Nucleotide sequence of SARS-CoV-2N Gene:
AUGUCUGAUAAUGGACCCCAAAAUCAGCGAAAUGCACCCCGCAUUACGUUUGGUGGACCCUCAGAUUCAACUGGCAGUAACCAGAAUGGAGAACGCAGUGGGGCGCGAUCAAAACAACGUCGGCCCCAAGGUUUACCCAAUAAUACUGCGUCUUGGUUCACCGCUCUCACUCAACAUGGCAAGGAAGACCUUAAAUUCCCUCGAGGACAAGGCGUUCCAAUUAACACCAAUAGCAGUCCAGAUGACCAAAUUGGCUACUACCGAAGAGCUACCAGACGAAUUCGUGGUGGUGACGGUAAAAUGAAAGAUCUCAGUCCAAGAUGGUAUUUCUACUACCUAGGAACUGGGCCAGAAGCUGGACUUCCCUAUGGUGCUAACAAAGACGGCAUCAUAUGGGUUGCAACUGAGGGAGCCUUGAAUACACCAAAAGAUCACAUUGGCACCCGCAAUCCUGCUAACAAUGCUGCAAUCGUGCUACAACUUCCUCAAGGAACAACAUUGCCAAAAGGCUUCUACGCAGAAGGGAGCAGAGGCGGCAGUCAAGCCUCUUCUCGUUCCUCAUCACGUAGUCGCAACAGUUCAAGAAAUUCAACUCCAGGCAGCAGUAGGGGAACUUCUCCUGCUAGAAUGGCUGGCAAUGGCGGUGAUGCUGCUCUUGCUUUGCUGCUGCUUGACAGAUUGAACCAGCUUGAGAGCAAAAUGUCUGGUAAAGGCCAACAACAACAAGGCCAAACUGUCACUAAGAAAUCUGCUGCUGAGGCUUCUAAGAAGCCUCGGCAAAAACGUACUGCCACUAAAGCAUACAAUGUAACACAAGCUUUCGGCAGACGUGGUCCAGAACAAACCCAAGGAAAUUUUGGGGACCAGGAACUAAUCAGACAAGGAACUGAUUACAAACAUUGGCCGCAAAUUGCACAAUUUGCCCCCAGCGCUUCAGCGUUCUUCGGAAUGUCGCGCAUUGGCAUGGAAGUCACACCUUCGGGAACGUGGUUGACCUACACAGGUGCCAUCAAAUUGGAUGACAAAGAUCCAAAUUUCAAAGAUCAAGUCAUUUUGCUGAAUAAGCAUAUUGACGCAUACAAAACAUUCCCACCAACAGAGCCUAAAAAGGACAAAAAGAAGAAGGCUGAUGAAACUCAAGCCUUACCGCAGAGACAGAAGAAACAGCAAACUGUGACUCUUCUUCCUGCUGCAGAUUUGGAUGAUUUCUCCAAACAAUUGCAACAAUCCAUGAGCAGUGCUGACUCAACUCAGGCCUAA(SEQ ID NO:1)
the information on the designed crRNA nucleic acid sequence is shown in Table 1.
TABLE 1 CrRNA sequence of targeting N Gene
| CrRNA numbering | Nucleotide sequence of crRNA spacer (5 'to 3') | SEQ ID NO: |
| crRNA 2 | CUCUCAAGCUGGUUCAAUCUGUCA | 2 |
| crRNA 3 | GUCAUCUGGACUGCUAUUGG | 3 |
| crRNA 4 | UUUGCGGCCAAUGUUUGUAA | 4 |
| crRNA 6 | CCAGACAUUUUGCUCUCAAG | 5 |
| crRNA 12 | UCUGGUUACUGCCAGUUGAA | 6 |
| crRNA 13 | GGAUUGCGGGUGCCAAUGUG | 7 |
| crRNA 14 | UGUAGCACGAUUGCAGCAUU | 8 |
| crRNA 16 | GAAGCGCUGGGGGCAAAUUG | 9 |
| crRNA 17 | AUGCGCGACAUUCCGAAGAA | 10 |
| crRNA 18 | UUGGUGUAUUCAAGGCUCCC | 11 |
(2) Preparation of crRNA molecules by in vitro transcription
The crRNA molecules of table 1 were prepared by in vitro transcription using SP6 RNA polymerase (manufacturer New England Biolabs).
Further, the in vitro transcribed product was purified using RNA beads (manufacturer Vazyme).
(3) RT-qPCR, cas13 detection SARS-CoV-2N target sequence and degradation sequence thereof
Cas13 detection system as shown in table 2, the fluorescence value of the Cas13 detection system reaction product was measured using a real-time quantitative PCR instrument using a 10 μl system. The reaction was carried out at 37℃and the fluorescence value was read every 30s for 2 hours.
The RT-qPCR detection system (manufacturer Vazyme) is shown in Table 3, and 10. Mu.L of the system is adopted, and a real-time quantitative PCR instrument is adopted to measure the reaction product of the RT-qPCR detection system. The reaction procedure is shown in Table 4.
The crRNA composition selection scheme is shown in fig. 3 and 4, and the nucleotide sequence information is shown in table 1.
TABLE 2 Cas13 detection System
| Composition of the components | Dosage of |
| 10×Cas13 reaction buffer | 1μL |
| LbuCas13a(8.6μM) | 0.115μL |
| crRNA pool(500nM) | 1μL |
| Single-stranded RNA fluorescent reporter (10. Mu.M) | 0.4μL |
| PEG 35,000(41％) | 0.4μL |
| Degraded or undegraded target RNA molecules | 1μL |
| RNase-free water | Up to 10μL |
Remarks: "crRNA pool" comprises single or multiple crRNA molecules, the nucleotide sequences of which are described in table 1. The sequence of the single-stranded RNA fluorescent reporter molecule is as follows: 5 'FAM-UUUUU-3' BHQ1.
TABLE 3 RT-qPCR detection System
| Composition of the components | Dosage of |
| 2×One Step Q Probe Mix | 5μL |
| One Step Q Probe Enzyme Mix | 0.5μL |
| Upstream primer (10. Mu.M) | 0.2μL |
| Downstream primer (10. Mu.M) | 0.2μL |
| Degraded or undegraded target RNA molecules | 1μL |
| RNase-free water | Up to 10μL |
TABLE 4 RT-qPCR reaction procedure
The detection results are shown in FIG. 1. 1) The RT-qPCR detection method (figure 1A) and the detection of degraded or undegraded target RNA molecules by using single or two crRNAs (1 and 2 in figure 1B) have great influence on the detection of the target RNA molecules by degradation, and the detection rate of the degraded target RNA molecules is obviously reduced.
2) As can be seen from fig. 1B, 3, 4 and 5, when 3 or more crrnas are used to detect degraded or undegraded target RNA molecules, the effect of degradation on the detection of target RNA molecules can be effectively eliminated.
3) As can be seen from FIG. 1B, 5 crRNAs were used to detect degraded or undegraded target RNA molecules, which could still be detected when the target RNA molecule concentration was as low as 50 fM.
The experimental result shows that the method can be used for detecting degraded RNA, and the initial target RNA concentration of a sample is low and the detection rate is high; the method of the invention not only can effectively eliminate the influence of degradation on target nucleic acid detection, but also can obviously improve the detection sensitivity.
Further, the inventors examined whether different crRNA combinations have higher detection rates and sensitivities for the degraded target RNA molecules. The detection results are shown in FIG. 2. When different crRNA combinations are used, the detection sensitivity of multiple crrnas is still higher than that of a single crRNA. When multiple crrnas are used to detect degraded or undegraded target RNA molecules, the effect of degradation on target RNA molecule detection is eliminated.
Example 2: the method of the present invention is used in detecting SARS-CoV-2N gene degraded seriously
In this example, severely degraded target RNA molecules were detected using the detection methods of the present invention and the conventional RT-qPCR detection methods, respectively.
In this example, the severely degraded SARS-CoV-2N gene (target RNA molecule) is obtained by the following procedure: the target RNA molecules are added into 0.1M NaOH solution and heated for 60-150 min at 95 ℃. The target RNA molecules can not be detected by adopting RT-qPCR to quantify the target RNA molecules of the degraded SARS-CoV-2N gene.
In this example, the RT-qPCR detection system (Santa Clara, inc.) is shown in Table 5, using a 10. Mu.L system and measuring the reaction products of the RT-qPCR detection system using a real-time quantitative PCR instrument. The reaction procedure is shown in Table 6.
The detection method of the present invention uses 5 crrnas for target nucleic acid detection, the combination of these 5 crrnas being: crRNA 14, crRNA 4, crRNA 2, crRNA 12 and crRNA 3 (for nucleotide sequence information of crRNA see table 1 of example 1), cas13 detection system is the same as example 1.
TABLE 5 RT-qPCR detection System
| Composition of the components | Dosage of |
| 2019-nCoV-PCR-reaction solution | 5.2μL |
| 2019-nCoV-PCR-enzyme mixed solution | 0.8μL |
| Degraded or undegraded target RNA molecules | 1μL |
| RNase-free water | Up to 10μL |
TABLE 6 RT-qPCR reaction procedure
The detection results are shown in FIG. 5. For severely degraded RNA molecules, target RNA molecules could not be detected using conventional RT-qPCR, but could be effectively detected using 5 crRNA molecules.
The experimental result shows that the method can be used for detecting the RNA which is seriously degraded, and the detection rate is high.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (13)
1. A crRNA composition for targeting a target nucleic acid, comprising: at least three crrnas;
Wherein each of the crrnas targets the same target nucleic acid;
any two recognition regions of the crRNA-targeted target nucleic acids do not overlap;
a spacing region exists between the recognition regions of two adjacent crrnas, and the spacing regions of the target nucleic acids corresponding to the crRNA compositions are not less than two.
2. The composition according to claim 1, characterized in that the recognition region of the crRNA is 17-28 nucleotides in length, preferably 19-28 nucleotides;
optionally, the spacer region is no less than 30 nucleotides in length, preferably 50 to 250 nucleotides, more preferably 100 to 200 nucleotides;
optionally, the sum of the lengths of the recognition region and the spacer region corresponding to the crRNA composition is 30-100% of the total length of the target nucleic acid;
optionally, the sum of the lengths of the recognition region and the spacer region corresponding to the crRNA composition is 50-100% of the total length of the target nucleic acid;
optionally, the sum of the lengths of the recognition and spacer regions corresponding to the crRNA composition is 60-100% of the total length of the target nucleic acid.
3. The composition according to claim 1, wherein the target nucleic acid is RNA, preferably RNA derived from RNA virus;
optionally, the target nucleic acid is the N gene of SARS-COV-2 virus; preferably, the target nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 1;
Optionally, the crRNA composition comprises at least three crRNAs and has at least three of the nucleotide sequences as shown in SEQ ID NOS.2-11; preferably, the crRNA composition is three crRNAs and has a nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 8;
optionally, the crRNA composition comprises at least four crRNAs and has at least four of the nucleotide sequences as shown in SEQ ID NOs 2-11; preferably, the crRNA composition is four crRNAs and has a nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8;
optionally, the crRNA composition comprises at least five crRNAs and has at least five of the nucleotide sequences as shown in SEQ ID NOS.2-11; preferably, the crRNA composition is five crRNAs and has nucleotide sequences shown as SEQ ID NO. 2-4, SEQ ID NO. 6 and SEQ ID NO. 8; preferably, the crRNA composition is five crRNAs and has nucleotide sequences shown as SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 9-11;
optionally, the crRNA composition is ten crRNAs and has a nucleotide sequence as shown in SEQ ID NO. 2-11.
4. A method of obtaining a crRNA composition for targeting a target nucleic acid, comprising:
Providing sequence information of the target nucleic acid;
designing a plurality of candidate crrnas targeting the target nucleic acid sequence based on sequence information of the target nucleic acid;
screening at least three crrnas meeting a condition from the plurality of candidate crrnas as target crrnas:
wherein the satisfaction condition includes:
each of the target crrnas targets the same target nucleic acid;
any two recognition regions of the target crRNA-targeting target nucleic acid do not overlap;
a spacing region exists between the recognition regions of two adjacent target crrnas, and the spacing regions of all target crrnas corresponding to the target nucleic acids are not less than two.
5. The method according to claim 4, wherein the recognition region of the target crRNA is 17 to 28 nucleotides in length, preferably 19 to 28 nucleotides;
optionally, the spacer region is no less than 30 nucleotides in length, preferably 50 to 250 nucleotides, more preferably 100 to 200 nucleotides;
optionally, the sum of the lengths of the recognition regions and the interval regions corresponding to all target crrnas is 30-100% of the total length of the target RNA sequence;
optionally, the sum of the lengths of the recognition regions and the interval regions corresponding to all target crrnas is 50-100% of the total length of the target RNA sequence;
Optionally, the sum of the lengths of the recognition and spacer regions corresponding to all target crrnas is 60-100% of the total length of the target RNA sequence.
6. The method according to claim 4, wherein the target nucleic acid is RNA, preferably RNA derived from RNA virus;
optionally, the target nucleic acid is the N gene of SARS-COV-2 virus; preferably, the target nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 1;
optionally, the crRNA composition obtained comprises at least three crRNAs and has at least three of the nucleotide sequences as shown in SEQ ID NOs 2 to 11; preferably, the crRNA composition obtained is three crRNAs and has a nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 8;
optionally, the crRNA composition obtained comprises at least four crRNAs and has at least four of the nucleotide sequences as shown in SEQ ID NOs 2 to 11; preferably, the crRNA composition obtained is four crRNAs and has a nucleotide sequence as shown in SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8;
optionally, the crRNA composition obtained comprises at least five crRNAs and has at least five of the nucleotide sequences as shown in SEQ ID NOs 2 to 11; preferably, the obtained crRNA composition is five crRNAs and has nucleotide sequences shown as SEQ ID NO. 2-4, SEQ ID NO. 6 and SEQ ID NO. 8; preferably, the obtained crRNA composition is five crRNAs and has nucleotide sequences shown as SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 9-11;
Optionally, the target crRNA composition obtained is ten crRNAs and has the nucleotide sequence as shown in SEQ ID NOs 2 to 11.
7. The method of claim 4, wherein the designing a plurality of candidate crrnas that target a target nucleic acid sequence is performed using crRNA design software;
optionally, the crRNA design software is Cas13design.
8. A system for detecting a target nucleic acid in a sample, comprising:
a crRNA composition according to any one of claims 1 to 3 or obtainable according to the method of any one of claims 4 to 7;
optionally, further comprising: cas proteins and reporter molecules.
9. A composition for detecting a target nucleic acid in a sample, comprising:
a crRNA composition according to any one of claims 1 to 3 or obtainable according to the method of any one of claims 4 to 7;
optionally, further comprising: cas proteins and reporter molecules.
10. A kit for detecting a target nucleic acid in a sample, comprising: the composition for detecting a target nucleic acid in a sample of claim 9.
11. Use of the crRNA composition of any one of claims 1 to 3, the system of claim 8 or the composition of claim 9 in the preparation of a kit for detecting a target nucleic acid in a sample;
Optionally, the target nucleic acid is derived from a microorganism, soil, water source, animal or plant sample;
optionally, the target nucleic acid is derived from a virus or a bacterium;
optionally, the target nucleic acid comprises at least one of RNA, ASO, and a nucleic acid aptamer;
optionally, the target nucleic acid comprises at least one of mRNA, miRNA, and siRNA;
optionally, the target nucleic acid is derived from RNA of an RNA virus;
optionally, the target nucleic acid is the N gene of SARS-COV-2 virus;
preferably, the target nucleic acid has a nucleotide sequence as set forth in SEQ ID NO. 1.
12. A method for detecting a target nucleic acid in a sample, comprising:
step 1: determining sequence information of the target nucleic acid;
step 2: a crRNA composition obtained according to the method of any one of claims 4 to 7 or providing a crRNA composition of any one of claims 1 to 3;
step 3: reacting the crRNA composition, cas protein, and a reporter molecule for generating a detectable signal, collecting the detectable signal;
step 4: based on the detectable signal, the target nucleic acid is qualitatively or quantitatively analyzed.
13. The method of claim 12, wherein step 3 comprises: a reaction system is constructed based on the system of claim 8 or the composition of claim 9 or the kit of claim 10, and a reaction is performed to collect a detectable signal.
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