CN111662968A - Detection method of PAM-sequence-free DNA based on CRISPR and application thereof - Google Patents
Detection method of PAM-sequence-free DNA based on CRISPR and application thereof Download PDFInfo
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Abstract
The invention provides a detection method and application of PAM-sequence-free DNA based on CRISPR, wherein the detection system comprises an asymmetric PCR amplification system and a CRISPR-Cas detection system; the asymmetric PCR amplification system comprises a forward and a reverse specific primer pair. According to the invention, the accumulation of single-stranded DNA in a PCR system is controlled by adjusting the proportion of the forward/reverse primers, the cis-form cutting activity of the CRISPR-Cas system is activated by using the generated reverse single-stranded DNA, the detection of the target double-stranded DNA is carried out, the restriction effect of a PAM sequence on the traditional CRISPR-Cas detection system is eliminated, and the application range of the CRISPR-Cas detection system is expanded.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, belongs to the technical field of nucleic acid detection, and relates to a detection method of DNA without a PAM sequence based on CRISPR and application thereof.
Background
Jennifer Doudna discovered in 2018, as shown in FIG. 1, when Cas12a protein, gRNA and target double-stranded DNA (target dsDNA) form a ternary complex structure, Cas12a protein not only cleaves target dsDNA in trans, but also cleaves nearby short-chain probe DNA (Probe ssDNA) in cis without difference, so that the short-chain probe DNA modified with a fluorophore and a quencher at both ends is designed, when the short-chain probe DNA is cleaved, fluorescence is generated, and the fluorescence intensity is proportional to the concentration of the target DNA within a certain time, and the target DNA can be subjected to rapid real-time fluorescence detection by using the characteristics of the CRISPR system (Chen J S, Ma E, Harrington L B, CRISPR-Cas12a target binding and fluorescent single-stranded activity [ J201360, Science, 8, 6387) 439). The cutting characteristics of the CRISPR-Cas12a system on target single-stranded DNA and non-target single-stranded DNA were studied systematically by Zhao national screen team of Chinese academy of sciences in 2018, 3/12.T.once again, the unique advantages of the CRISPR-Cas12a system in the field of nucleic acid detection (Li S Y, Cheng Q X, Liu J K, et al. CRISPR-Cas12a has both cis-andtran-cleavage activity on single-stranded DNA [ J ]. Cell research,2018,28(4): 491-. Then, the Zhang FengO group realizes the rapid detection of trace target DNA on paper-based materials by using a CRISPR-Cas12a system based on recombinase polymerase isothermal amplification technology (RPA), and the sensitivity can reach the order of aM/L (Gootenberg J S, Abudayyeh O, Kellner M J, et al. multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6[ J ] Science,2018,360 (6387: 439 and 444.). Chen believes that CRISPR-Cas12a can be a powerful tool for DNA detection from various sources, and the detect (DNA Endonuclease Targeted CRISPR transreporter) technique is potentially applicable to any POCT detection based on DNA analysis, including cancer and infectious diseases.
CN 110541022A discloses a Mycobacterium tuberculosis complex detection kit based on a CRISPR-Cas12a system, which improves the detection sensitivity by adopting a recombinase polymerase amplification technology, activates the bypass cleavage activity of Cas12a after the CRISPR-Cas12a specifically targets a Mycobacterium tuberculosis complex target sequence, can sensitively and specifically detect the Mycobacterium tuberculosis complex from sputum, and has the advantages of no invasion, frequent multiple detection, high detection speed and the like.
CRISPR targeting specificity is determined by two parts, one part is base pairing between RNA chimera (crRNA) and target DNA, and the other part is interaction between Cas protein and a short DNA sequence (usually at the 3' end of target DNA), which is called Protospacer Adjacent Motif (PAM), therefore, the CRISPR-Cas12a system strongly depends on the PAM sequence in the target DNA, i.e. when the target DNA does not contain PAM sequence (TTTN), Cas12a protein in the CRISPR-Cas12a system cannot bind to target DNA through gRNA, cis-cleavage activity cannot be activated, and the detection system will fail, which greatly limits the applicability of the system.
Therefore, in order to expand the applicable range of the CRISPR-Cas12a system without being limited by the PAM sequence, the traditional CRISPR-Cas12a system needs to be improved.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior art, the invention provides a method for detecting DNA without a PAM sequence based on CRISPR and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides a CRISPR-based PAM-sequence-free DNA detection system, which comprises an asymmetric PCR amplification system and a CRISPR-Cas detection system;
the asymmetric PCR amplification system comprises a forward and a reverse specific primer pair.
According to the invention, by utilizing the principle that the CRISPR-Cas12a/b system is not limited by a PAM sequence in the detection of single-stranded DNA, excessive reverse primers of the target DNA are added in the PCR process to carry out asymmetric PCR, a large amount of reverse target single-stranded DNA matched with crRNA is generated, and the homeotropic cleavage activity of the CRISPR-Cas12a/b system can be activated after the reverse target single-stranded DNA is combined with the crRNA, so that the detection of the target double-stranded DNA is realized.
Preferably, the molar amount of the forward primer in the specific primer pair is less than that of the reverse primer, preferably the molar ratio of the forward primer to the reverse primer is 1 (2-50), and may be, for example, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:30, 1:40 or 1:50, preferably 1 (2-10).
In the invention, the molar ratio of the forward primer to the reverse primer is set to be 1 (2-50), so that not only is amplification of target double-stranded DNA completed, but also reverse target single-stranded DNA matched with crRNA can be generated, the homeotropic cleavage activity of a CRISPR-Cas12a/Cas12b system is activated, and the detection of the target double-stranded DNA is realized; if the molar ratio of the forward primer to the reverse primer is less than 1 (2-50), sufficient reverse target single-stranded DNA cannot be generated, and if the molar ratio of the forward primer to the reverse primer is more than 1 (2-50), amplification of the target double-stranded DNA cannot be completed.
Preferably, the asymmetric PCR amplification system further comprises a DNA polymerase, which may be, for example, LA Taq DNA polymerase (TAKaRa) or Pyrobest DNA polymerase (TAKaRa), dNTPs comprising dATP, dTTP, dCTP and dGTP, and a template.
Preferably, the CRISPR-Cas detection system comprises a Cas protein and a crRNA.
Preferably, the Cas protein comprises Cas12a, preferably any one or a combination of at least two of the equivalents AsCas12a, LbCas12a or FnCas12 a.
According to the present invention, the third gene scissors CRISPR-Cas12B developed by the recently Zhang Bow team has similar bypass cleavage activity to Cas12a protein (Strecker J, Jones S R, Koopal B, et al. engineering of CRISPR-Cas12B for human Genome editing [ J ]. Nature Communications,2019,10(1):212-212.), and the Liwei team has invented CDelection technology based on it for DNA detection and single base analysis (Teng F, Guo L, Cui T, et al. CDelection: CRISPR-Cas12B-based DNA detection with-interaction sensitivity and single-base specificity [ J ]. Genome Biology,2019,20 (1)). Based on the above references, Cas12b has the same biological activity as Cas12a, and can also achieve the effect of DNA cleavage, and the invention adopts Cas protein comprising Cas12b, preferably any one or a combination of at least two of the equivalent sources of aa Cas12b, BhCas12b or AkCas12 b. However, Cas12b is characterized by a smaller size and lower off-target rate than Cas12a, but since it is derived from thermotolerant acidophilic bacteria, the optimal active temperature is as high as 48 ℃, so it is clear that low temperature Cas12a has certain advantages in the field of rapid detection.
In the invention, the single-stranded DNA cleavage activity of the Cas12a/b protein is utilized, which is beneficial to realizing DNA detection independent of PAM sequence.
Preferably, the CRISPR-Cas detection system further comprises a probe.
Preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group.
In the invention, the fluorescence probe is cut by using the bypass cutting activity of the Cas12a/b protein, so that the real-time fluorescence detection of the target DNA is realized.
In a second aspect, the present invention provides a CRISPR-based method for detecting PAM-free sequence DNA, comprising the steps of:
(1) carrying out asymmetric PCR by adopting a specific primer pair of the target DNA;
(2) and detecting the amplification product by using a CRISPR-Cas detection system.
Preferably, the molar amount of the forward primer in the specific primer pair is less than that of the reverse primer, and preferably, the molar ratio of the forward primer to the reverse primer is 1 (2-50).
Preferably, the conditions of the asymmetric PCR are that denaturation is carried out for 30-40 s at the temperature of 93-95 ℃, annealing is carried out for 10-30 s at the temperature of 50-60 ℃, extension is carried out for 20-30 s at the temperature of 70-72 ℃, and circulation is carried out for 30-40 times.
Preferably, the CRISPR-Cas detection system contains a probe.
Preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group.
In a third aspect, the invention provides a CRISPR-based CYP1A1 detection kit, which comprises a forward primer shown as SEQ ID NO. 1 and a reverse primer shown as SEQ ID NO. 2;
SEQ ID NO:1：5’-AAGAGAAAGACCTCCCAGCGGGCAA-3’；
SEQ ID NO:2：5’-CACCCCTGATGGTGCTATCGACAAG-3’。
in the invention, the specific primer pair of CYP1A1 is designed according to the sense strand and the antisense strand of CYP1A1, wherein the sense strand of CYP1A1 is shown as SEQ ID NO. 5, and the antisense strand is shown as SEQ ID NO. 6;
SEQ ID NO:5：
5’-AAGAGAAAGACCTCCCAGCGGGCAATGGTCTCACCGATACACTTCCGCTTGCCCATGCCAAAGATAATCACCTTCTCACTTAACACCTTGTCGATAGCACCATCAGGGGTG-3’；
SEQ ID NO:6：
5’-CACCCCTGATGGTGCTATCGACAAGGTGTTAAGTGAGAAGGTGATTATCTTTGGCATGGGCAAGCGGAAGTGTATCGGTGAGACCATTGCCCGCTGGGAGGTCTTTCTCTT-3’。
preferably, the molar ratio of the forward primer to the reverse primer is 1 (2-50).
Preferably, the kit further comprises a DNA polymerase and dNTPs.
Preferably, the kit further comprises a Cas protein and a crRNA.
Preferably, the Cas protein comprises Cas12a, preferably any one or a combination of at least two of the equivalents AsCas12a, LbCas12a or FnCas12 a.
Preferably, the Cas protein comprises Cas12b, preferably any one or a combination of at least two of AaCas12b, BhCas12b or AkCas12b equivalent homologues.
Preferably, the crRNA is shown as SEQ ID NO. 3;
SEQ ID NO:3：
5’-UAAUUUCUACUCUUGUAGAUCAAUGGUCUCACCGAUACAC-3’。
preferably, the kit further comprises a probe.
Preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group.
Preferably, the probe is shown as SEQ ID NO. 4;
SEQ ID NO:4：5’-FAM-TTATT-BHQ1-3’。
in a fourth aspect, the present invention provides a use of the detection system of the first aspect for detecting DNA without PAM sequence.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the invention, by using the principle that the detection of the CRISPR-Cas12a/b system on the single-stranded DNA is not limited by a PAM sequence, an excessive reverse primer of the target DNA is added in the PCR process to carry out asymmetric PCR, so that a large amount of reverse target single-stranded DNA matched with the crRNA is generated, and the homeotropic cleavage activity of the CRISPR-Cas12a system or the CRISPR-Cas12b system can be activated after the reverse target single-stranded DNA is combined with the crRNA, so that the detection of the target double-stranded DNA is realized;
(2) according to the invention, the molar ratio of the forward primer to the reverse primer is set to 1 (2-50), so that not only is amplification of a target double-stranded DNA completed, but also a large amount of reverse target single-stranded DNA matched with crRNA can be generated, the homeotropic cleavage activity of a CRISPR-Cas12a system or a CRISPR-Cas12b system is activated, the detection of the target double-stranded DNA is realized, and the application range of the CRISPR-Cas12 detection system is expanded.
Drawings
FIG. 1 is a schematic diagram of a CRISPR-Cas12a detection system;
FIG. 2 is a gel electrophoresis of the asymmetric PCR amplification product, wherein the first lane shows DNA molecular weight (25-500 bp), the second lane shows the amplification product with excessive forward primer, and the third lane shows the amplification product with excessive reverse primer;
FIG. 3 is a real-time fluorescence curve of the amplification product.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Example 1 asymmetric PCR amplification of CYP1A1 Gene
In the embodiment, CYP1A1 gene is taken as an example, an excessive reverse primer is adopted to carry out asymmetric PCR, the system is shown in Table 1, and a control experiment with excessive forward primers is set, wherein the forward primer is shown as SEQ ID NO. 1, the reverse primer is shown as SEQ ID NO. 2, and the template is shown as SEQ ID NO. 5-6;
TABLE 1 asymmetric PCR detection System
| |
① Forward primer excess (2:1) | ② reverse primer excess (1:2) |
| Pyrobest(5U/μL) | 1.2μL | 1.2μL |
| 10×Buffer | 10μL | 10μL |
| ddH2O | 78μL | 78μL |
| dNTP(2.5mM) | 9.6μL | 9.6μL |
| Forward primer (50. mu.M) | 2.4μL | 1.2μL |
| Reverse primer (50. mu.M) | 1.2μL | 2.4μL |
| Stencil (1 ng/. mu.L) | 1.2μL | 1.2μL |
Nucleic acid amplification is carried out by adopting a Veriti PCR amplification instrument of Thermo Fisher Science company under the conditions of denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s and extension at 72 ℃ for 20s, and the cycle is carried out for 40 times; and carrying out agarose gel electrophoresis detection on the amplification product.
FIG. 2 shows a gel electrophoresis image of the amplification product, wherein the first lane is the DNA molecular weight (25-500 bp from bottom to top), the second lane is the amplification product with excess forward primer (forward primer: reverse primer: 2:1), and the third lane is the amplification product with excess reverse primer (forward primer: reverse primer: 1: 2).
Example 2
In this example, asymmetric PCR was carried out using an excess amount of reverse primer based on CYP1A1 gene in example 1, and the primer sequence, template sequence, amplification conditions, etc. used are the same as those in example 1, as shown in Table 2;
TABLE 2 asymmetric PCR detection System
Example 3 CRISPR-Cas12a detection of amplified products
The real-time fluorescence detection of the amplification products of the embodiments 1 and 2 is performed based on a CRISPR-Cas12a detection system, wherein the system is shown in Table 3, crRNA is shown in SEQ ID NO. 3, and a probe is shown in SEQ ID NO. 4;
TABLE 3 CRISPR-Cas12a detection System
| Components | Dosage of |
| Cas12a protein (1.0. mu.M) | 1.25μL |
| crRNA(5.0μM) | 2.5μL |
| Probe needle | 2.5μL |
| 10×Buffer | 5μL |
| ddH2O | 15μL |
| ①, ②, ③, ④ or ⑤ | 25μL |
Real-time fluorescent quantitative detection is carried out by using a Spark enzyme-linked immunosorbent assay (ELIASA) of ENCO company.
As shown in fig. 3, which is a real-time fluorescence curve of an amplification product, it can be seen that only when the reverse primer is excessive and the PCR amplification product contains a large amount of reverse target single-stranded DNA, the CRISPR-Cas12a detection system is activated, and the Cas12a protein cleaves the fluorescent probe, thereby realizing real-time detection of the target dsDNA; with the increase of the proportion of the reverse primer to the forward primer, the signal appears more quickly, and the obvious difference appears only in 10-30 min.
In conclusion, the invention controls the accumulation of single-stranded DNA in a PCR system by regulating the proportion of forward/reverse primers, utilizes the generated reverse single-stranded DNA to activate the cis-cleavage activity of the CRISPR-Cas12a system, detects the target double-stranded DNA, eliminates the restriction of a PAM sequence on the traditional CRISPR-Cas12a detection system, and enlarges the application range of the CRISPR-Cas12a detection system.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
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Claims (10)
1. A PAM-sequence-free DNA detection system based on CRISPR is characterized by comprising an asymmetric PCR amplification system and a CRISPR-Cas detection system;
the asymmetric PCR amplification system comprises a forward and a reverse specific primer pair;
the molar amount of the forward primer in the specific primer pair is less than that of the reverse primer.
2. The detection system according to claim 1, wherein the asymmetric PCR amplification system further comprises DNA polymerase, dNTPs, and a template.
3. The detection system according to claim 1 or 2, wherein the CRISPR-Cas detection system comprises a Cas protein and a crRNA;
preferably, the Cas protein comprises Cas12a and/or Cas12 b;
preferably, the Cas protein comprises any one of AsCas12a, LbCas12a, FnCas12a, AaCas12b, BhCas12b or AkCas12b, or a combination of at least two thereof;
preferably, the CRISPR-Cas detection system further comprises a probe DNA;
preferably, the 5 'end of the probe DNA is modified with a fluorescent group, and the 3' end of the probe DNA is modified with a quenching group.
4. A method for detecting PAM-free sequence DNA based on CRISPR (clustered regularly interspaced short palindromic repeats), which is characterized by comprising the following steps:
(1) carrying out asymmetric PCR by adopting a specific primer pair of the target DNA;
(2) and detecting the amplification product by using a CRISPR-Cas detection system.
5. The detection method according to claim 4, wherein the specific primer pair has a smaller molar amount of forward primer than reverse primer;
preferably, the conditions of the asymmetric PCR are that denaturation is carried out for 30-40 s at the temperature of 93-95 ℃, annealing is carried out for 10-30 s at the temperature of 50-60 ℃, extension is carried out for 20-30 s at the temperature of 70-72 ℃, and circulation is carried out for 30-40 times.
6. The detection method according to claim 4 or 5, wherein the CRISPR-Cas detection system contains probe DNA;
preferably, the 5 'end of the probe DNA is modified with a fluorescent group, and the 3' end of the probe DNA is modified with a quenching group.
7. A CYP1A1 detection kit based on CRISPR is characterized by comprising a forward primer shown as SEQ ID NO. 1 and a reverse primer shown as SEQ ID NO. 2;
preferably, the molar ratio of the forward primer to the reverse primer is 1 (2-50);
preferably, the kit further comprises a DNA polymerase and dNTPs.
8. The kit of claim 7, further comprising a Cas protein and a crRNA;
preferably, the Cas protein comprises Cas12a and/or Cas12 b;
preferably, the Cas protein comprises any one of AsCas12a, LbCas12a, FnCas12a, AaCas12b, BhCas12b or AkCas12b, or a combination of at least two thereof;
preferably, the crRNA is shown as SEQ ID NO 3.
9. The kit of claim 7 or 8, wherein the kit further comprises a probe;
preferably, the 5 'end of the probe is modified with a fluorescent group, and the 3' end of the probe is modified with a quenching group;
preferably, the probe is shown as SEQ ID NO. 4.
10. Use of a detection system according to any one of claims 1 to 3 for the detection of DNA free of PAM sequences.
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