CN113174433A

CN113174433A - Cas protein-based detection method

Info

Publication number: CN113174433A
Application number: CN202110436508.6A
Authority: CN
Inventors: 梁德生; 张春华; 周妙金; 邬玲仟
Original assignee: Central South University
Current assignee: Suzhou Ganjiang Biotechnology Co ltd
Priority date: 2021-04-22
Filing date: 2021-04-22
Publication date: 2021-07-27
Anticipated expiration: 2041-04-22
Also published as: CN113174433B

Abstract

The invention relates to a detection method based on a Cas protein, which comprises the following steps: s1, designing and synthesizing a PCR amplification primer according to the target gene; synthesizing crRNA according to the PCR amplification primer; s2, carrying out PCR amplification on the target gene by adopting a PCR amplification primer to obtain an amplification product; then, detecting an amplification product by using a Cas protein, crRNA and fluorescent probe system; the sequences of the primers comprise a PAM sequence and a crRNA recognition region sequence. The invention breaks through the limits of the PAM sequence and the crRNA, and can also artificially introduce the PAM sequence and the crRNA through the primer for detection even under the condition that no PAM sequence exists near the mutation site to be detected or the proper crRNA cannot be designed. And the primer-mediated Cas12a qualitative detection can detect a plurality of mutation sites in 1 reaction system, embodies the capability of multiple detection, is beneficial to reducing operation steps and reducing detection cost.

Description

Cas protein-based detection method

Technical Field

The invention relates to a nucleic acid molecule detection method based on a Cas protein.

Background

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated nucleases (CRISPR associated proteins, Cas) commonly present in bacteria and archaea are defense systems of bacteria against foreign plasmid or phage infection, and scientists have discovered a variety of Cas proteins and their working mechanisms one after another. Cas12a (also known as Cpf1) specifically recognizes and is activated by double stranded DNA (dsDNA) under crRNA guidance, and activated Cas12a can specifically cleave target dsDNA molecules and non-specifically cleave unrelated single stranded DNA (ssDNA), i.e., exhibits cleavage activity.

Detection of target dsDNA molecules using the attendant cleavage activity of Cas12a, principle of prior art protocol: designing a primer according to a target dsDNA molecule sequence to be detected, amplifying the target dsDNA molecule by a PCR or isothermal amplification method, if the target dsDNA molecule exists, generating an amplification product of the target dsDNA molecule in an amplification system, designing and synthesizing crRNA according to the target dsDNA molecule sequence to be detected, guiding the crRNA to Cas12a to identify the target dsDNA molecule, taking a reaction solution of the amplification system, incubating the reaction solution with Cas12a, the crRNA and a probe ssDNA (fluorescent probe), and detecting whether a fluorescent signal exists or not through an instrument after the incubation is finished to judge whether the Cas12a is activated or not, thereby qualitatively detecting the target dsDNA molecule.

Chen et al combines Cas12a with recombinase polymerase isothermal amplification (RPA) technology, Li et al combines Cas12a with PCR technology, and develops Cas12 a-based nucleic acid detection technologies DETECTOR and HOLMES, respectively, the basic principle is as follows: designing crRNA according to the target dsDNA, specifically recognizing and cutting the amplified target dsDNA molecule by a Cas12a-crRNA compound, simultaneously activating Cas12a, cutting the ssDNA probe by utilizing the attached cutting activity of the activated Cas12a, respectively marking a fluorescent group and a quenching group at two ends of the probe, releasing a fluorescent signal after the probe is cut, and further detecting the target dsDNA by reading a detection signal released after the probe is cut (figure 1)^[1,2]。

However, the nucleic acid detection technology based on Cas12a has the following defects:

PAM sequence restriction

The first precondition for Cas12a to recognize the target dsDNA is: the target dsDNA must have the corresponding PAM sequence TTTN or TTN present. Only when the PAM sequence is present, it is possible to design and detect crRNA 3' downstream of the PAM sequence (FIG. 2). If there is no PAM sequence in the target sequence, crRNA cannot be designed and thus cannot be detected with Cas12 a. The PAM sequence greatly limits the range of choice of target sequences and the flexibility of crRNA design.

Restriction of crRNA

A second precondition for Cas12a to recognize the target dsDNA is: the target dsDNA must have the appropriate crRNA recognition region sequence present. crRNA is a key molecule that directs Cas12a to recognize and determine the specificity of detection. Even if a PAM sequence is present in the target sequence to be detected, if a crRNA with high specificity cannot be designed downstream of PAM (fig. 2), the specificity of detection may be reduced, and thus detection may not be possible. Since Cas12a has some tolerance for base mismatch between crRNA and target sequence, it is also the reason why CRISPR/Cas system may be off-target in gene editing.

3. Limited to qualitative detection

Most of the existing Cas12a nucleic acid detection technologies are qualitative detection, and the qualitative detection can only judge whether a target nucleic acid molecule exists, but cannot judge the copy number of the target nucleic acid molecule. Only a few studies have conceptually achieved quantitative detection^[3,4]However, in practical applications, there are often concentration differences between samples, and in order to compare the differences in copy number of target nucleic acids between samples, an internal reference is also required to be established to eliminate the differences in quantitative detection results caused by the differences in sample concentration.

4. Confined to pathogen detection

Pathogen genome sequence is relatively large, and a suitable PAM sequence is expected to be found and a crRNA with high specificity is designed, so that a plurality of literature reports exist at present for applying Cas12a nucleic acid detection technology to Zika virus and dengue virus^[5]Novel coronavirus pneumovirus^[6,7]And Mycobacterium tuberculosis^[8]And the like, and the qualitative detection of various pathogens.

Common types of variation in genetic diseases include point mutations, small insertions or deletions. For point mutations, small insertions or deletions, the variation range is limited to 1 or several bases, and the PAM sequence is often absent nearby or suitable crRNA is difficult to design, so that the detection is difficult by the existing Cas12a nucleic acid detection technology. Beta-thalassemia is caused mainly by point mutations in the gene encoding globin.

Thalassemia (thalassemia) is caused by a variation in the gene encoding globin, a reduction or complete loss of globin synthesis, resulting in an imbalance in the globin chain and thus genetic hemolytic anemia. The carrying rate and incidence rate of alpha-thalassemia and beta-thalassemia in people in southern areas of China are high. Thalassemia patients are clinically manifested by hemolytic anemia, developmental retardation, specific facial appearance and hepatosplenomegaly, while carriers usually have no obvious clinical symptoms, or are only manifested by alterations in hematological indices, such as a decrease in Mean Corpuscular Volume (MCV), a decrease in Mean Corpuscular Hemoglobin (MCH), and the appearance of abnormal hemoglobin.

At present, the domestic beta-thalassemia carrier screening mainly adopts a screening strategy based on hematology phenotype, and the MCV, MCH reduction and HbA2 increase are positive indexes of the beta-thalassemia carrier. The carriers are screened out and then subjected to molecular detection to determine the genotypes of the carriers, but the screening strategy may omit the carriers with negative hematological phenotypes and also may misjudge the individuals with iron-deficiency anemia as the carriers.

Since the mutation type of the beta-thalassemia is mainly point mutation, the mutation is limited to 1 or a few bases, a PAM sequence is often absent nearby or a proper crRNA is difficult to design, and thus the mutation type is difficult to detect by using the existing Cas12a nucleic acid detection technology.

Reference to the literature

[1]CHEN J S,MA E,HARRINGTON L B,et al.,CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity[J].Science,2018,360:436-439.

[2]LI S Y,CHENG Q X,WANG J M,et al.,CRISPR-Cas12a-assisted nucleic acid detection[J].Cell discovery,2018,4:20.

[3]LI L,LI S,WU N,et al.,HOLMESv2:A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation[J].ACS synthetic biology,2019,8:2228-2237.

[4]LI H,LI M,YANG Y,et al.,Aptamer-Linked CRISPR/Cas12a-Based Immunoassay[J].Anal. Chem.,2021,93:3209-3216.

[5]CURTI L A,PEREYRA-BONNET F,REPIZO G D,et al.,CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a(Cpf1)effector nuclease[J].Emerging microbes&infections,2020,9:1140-1148.

[6]BROUGHTON J P,DENG X,YU G,et al.,CRISPR-Cas12-based detection of SARS-CoV-2[J]. Nat.Biotechnol.,2020.

[7]KELLNER M J,KOOB J G,GOOTENBERG J S,et al.,SHERLOCK:nucleic acid detection with CRISPR nucleases[J].Nat.Protoc.,2019,14:2986-3012.

[8]AI J W,ZHOU X,XU T,et al.,CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis[J].Emerging microbes&infections,2019,8:1361-1369.

[9]KLEINSTIVER B P,TSAI S Q,PREW M S,et al.,Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells[J].Nat.Biotechnol.,2016,34:869-874.

[10]KIM D,KIM J,HUR J K,et al.,Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells[J].Nat.Biotechnol.,2016,34:863-868.

Disclosure of Invention

The invention aims to artificially introduce a PAM sequence and a crRNA recognition region through a primer-mediated strategy, and does not depend on the PAM sequence and the crRNA recognition region on target dsDNA, so that the target dsDNA can be detected even if no PAM sequence exists on the target dsDNA or specific crRNA is difficult to design.

In order to solve the technical problems, the technical scheme of the invention is as follows:

a Cas protein-based detection method, comprising the steps of:

s1, designing and synthesizing a PCR amplification primer according to the target gene; synthesizing crRNA according to the PCR amplification primer;

s2, carrying out PCR amplification on the target gene by adopting a PCR amplification primer to obtain an amplification product; then, detecting an amplification product by using a Cas protein, crRNA and fluorescent probe system;

the sequences of the primers comprise a PAM sequence and a crRNA recognition region sequence.

Preferably, the Cas protein is Cas12a, Cas13, Cas12b or Cas 14.

Further preferably, the Cas protein is Cas12 a.

Cas12a and Cas12b can recognize double-stranded DNA directly, the target nucleic acid molecule recognized by Cas13 is single-stranded RNA, and the target nucleic acid molecule recognized by Cas14 is single-stranded DNA. The target nucleic acid molecule in the application examples is double-stranded DNA and can be directly recognized by Cas12a or Cas12 b. If Cas13 is used, a detection step is added to transcribe a single-stranded RNA molecule from the target double-stranded DNA molecule for detection. If Cas14 is used, a detection step is added, and the detection can be performed after 1 strand of the target double-stranded DNA molecule is degraded to form a single-stranded DNA molecule.

The invention claims a PCR amplification primer, which comprises a PAM sequence and a crRNA recognition region sequence.

Preferably, the PAM sequence is TTTN (SEQ ID NO.1) or TTN (SEQ ID NO. 2); preferably, the crRNA recognition region sequence comprises 18-23 bases.

Preferably, the base at the 3' end of one of the amplification primers is a base modified by a locked nucleic acid.

The locked nucleic acid modification can not only increase the annealing temperature, but also improve the specificity.

Taking the HBB-28 mutation site as an example, the sequence of one of the amplification primer species, such as F, is designed according to the sequence of the HBB c. -78A > G mutation site, the normal base of the site is A, and G is obtained after mutation. In order to ensure the specificity of detection, the amplification primer F needs to specifically recognize the combination of the mutated sequence and the mutation primer F in the PCR process so as to amplify a product, but cannot combine with the normal sequence and amplify the product. Since F has no mismatch with the mutant sequence and F has 1 mismatch with the normal sequence, F anneals to the mutant sequence at a higher temperature than F anneals to the normal sequence. Theoretically, by utilizing the difference of annealing temperatures, the amplification primers F and R can amplify mutant sequences at higher annealing temperatures, but can not amplify normal sequences. In order to further increase the annealing temperature and improve the specificity of the amplified mutation site, the 3' end base of F is modified by locked nucleic acid, and the annealing temperature of the primer is increased by 3-8 ℃ after the locked nucleic acid is modified. Higher annealing temperatures can be set during PCR to specifically amplify sequences containing the mutation sites, but not to amplify normal sequences that are not mutated.

The invention claims the application of the PCR amplification primer in preparing a reagent for detecting beta-thalassemia.

The invention claims a kit, which comprises the PCR amplification primer.

The invention claims application of the kit in preparation of a reagent for detecting beta-thalassemia.

The invention claims a crRNA used for identifying PCR amplification primers.

The invention claims application of the crRNA in preparation of a reagent for detecting beta-thalassemia.

The invention claims a kit comprising the crRNA.

The invention claims a kit, which comprises the PCR amplification primer and crRNA for identifying the PCR amplification primer.

Preferably, the target gene is human beta-globin gene (HBB) and mutation sites thereof HBB: c. -78A > G (-28), HBB: c.126_129delCTTT (CD41-42) or HBB: c.316-197C > T (IVS-II-654).

The PCR amplification primers are shown in Table 1:

TABLE 1 primer, Probe and crRNA sequence information

The invention claims a kit, which comprises PCR amplification primers shown as SEQ ID NO.3-8 and crRNA shown as SEQ ID NO. 10.

The applications may all be for non-disease diagnostic purposes.

Since the additional cleavage activity of the Cas12a cleavage probe is non-specific, the probe sequence is only single-stranded DNA, and there is no special requirement.

Preferably, the PCR amplification reaction system is:

wherein, the mixed primer F comprises:

HBB-28 primer F10. mu.M

HBB CD41-42 primer F10. mu.M

HBB IVS-II-654 primer F10. mu.M

The mixed primer R comprises:

HBB-28 primer R10. mu.M

HBB CD41-42 primer R10. mu.M

HBB IVS-II-654 primer R10. mu.M

The amplification primers of 3 mutation sites are mixed together, so that 3 mutation sites can be simultaneously detected in one reaction system, and multiple detection is realized. After the collective detection (3 mutation sites are simultaneously detected) of the examinee, whether the examinee carries the 3 mutation sites can be determined. The technical advantages are as follows: multiple detection is carried out, 3 mutation sites can be screened in 1 detection, and the cost-benefit of the detection is improved; secondly, a plurality of body variation sites are detected in a gathering mode, and the method is suitable for the requirement of group screening.

Preferably, the PCR reaction conditions are shown in FIG. 6.

Preferably, the reaction system for detecting the sample by using the Cas12a fluorescent probe system is as follows:

the method can distinguish normal people from carriers on a plurality of mutation sites, is suitable for detecting whether an individual carries beta-thalassemia mutation, and further detects a single site if specific mutation sites need to be determined after the individual is detected as a carrier.

Preferably, the mutation site detection is performed in 3 groups, each group being amplified with a corresponding PCR primer.

Preferably, the PCR amplification system is as follows:

preferably, the PCR reaction conditions are shown in FIG. 6.

The invention is further explained below:

3 common mutations of beta-thalassemia in Chinese population HBB: c. -78A > G (-28), HBB: c.126-129 delCTTT (CD41-42) and HBB: c.316-197C > T (IVS-II-654) are taken as application examples, and the 3 mutation sites cannot be detected by the existing Cas12a nucleic acid detection technology. By using the online crRNA design software, the crRNA cannot be designed at the HBB: c. -78A > G mutation site, and the crRNA can be designed at the HBB: c.126-129 delCTTT and HBB: c.316-197C > T mutation sites, but the designed crRNA has poor specificity, has off-target effect and cannot be detected.

For example, when HBB is mutated at the ratio of c, 78A and G (-28), normal people do not have mutation, the 3' end base of HBB-28 primer F is G modified by locked nucleic acid, and can not be completely complementary with HBB gene, and the locked nucleic acid modification can increase annealing temperature, inhibit HBB-28 primer F from annealing with wild type HBB gene to form double chain, and theoretically can not amplify products. 28 mutation carrier, HBB-28 primer F/R and mutant HBB gene are completely complementary, and the product can be amplified by PCR. The amplification product is dsDNA, which contains PAM sequence and universal crRNA recognition region sequence in addition to the corresponding sequence of HBB gene, and is complementary with crRNA. Thus, Cas12a can recognize the amplification product and be activated under the guidance of crRNA, and the activated Cas12a cleaves the fluorescent probe and releases a fluorescent signal. The presence or absence of the mutation at the site can be detected by detecting the presence or absence of a fluorescent signal (FIG. 3).

Compared with the prior art, the invention has the following beneficial effects:

the method breaks through the limits of a PAM sequence and crRNA, and can also artificially introduce the PAM sequence and the crRNA through a primer for detection even under the condition that no PAM sequence exists near a mutation site to be detected or a proper crRNA cannot be designed.

Secondly, the primer-mediated Cas12a qualitative detection can detect a plurality of variable sites in 1 reaction system, embodies the capability of multiple detection, is beneficial to reducing operation steps and reduces the detection cost.

Drawings

FIG. 1 is a schematic diagram of a prior art Cas12 a-based nucleic acid detection technology;

FIG. 2 is a schematic diagram of the PAM sequence and the crRNA recognition region;

FIG. 3 is a schematic diagram of the primer-mediated qualitative detection principle of Cas12a according to the present invention;

FIG. 4 shows PCR reaction conditions for 4 annealing temperatures for the present invention;

FIG. 5 is a graph showing the results of experiments conducted to find suitable annealing temperatures for the present invention;

FIG. 6 shows the PCR reaction conditions established in the present invention

FIG. 7 shows the primer-mediated qualitative determination of Cas12a in normal humans and beta-thalassemia carriers according to the present invention;

FIG. 8 is a design schematic diagram of detecting PAM sequences and crRNAs near 3 mutation sites of HBB gene by using existing Cas12a detection method.

Detailed Description

The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.

Example 1

(1) According to the human HBB gene sequence, 3 common mutation sites HBB: c. -78A > G (-28), HBB: c.126_129delCTTT (CD41-42) and HBB: c.316-197C > T (IVS-II-654) of Chinese population are selected, and PCR amplification primers are designed and synthesized, as shown in Table 1.

TABLE 1 primer, Probe and crRNA sequence information

(2) Verifying the primers and groping the annealing temperature.

Firstly, HBB-28 primers F and R are taken as an example for explanation, 1 part of each of negative control (water), HBB: c. -78A > G (-28) carrier and normal human gDNA sample is selected, and each sample is respectively matched with 4 PCR amplification systems.

The PCR amplification system is as follows:

② PCR reaction conditions are approximately as shown in FIG. 4, with the changes that annealing temperatures are set to 63, 64, 65 and 66 ℃ respectively, annealing times are all 30s, and the rest conditions are the same. Each sample was amplified under 4 different reaction conditions, and the amplified products were subjected to agarose gel electrophoresis.

And thirdly, the electrophoresis result is shown in figure 5, and no obvious band exists between the negative control (water) and the amplification product of a normal person under the condition of 4 annealing temperatures after electrophoresis, which shows that the specificity of the primer is better, and a non-specific product is not amplified. The HBB c. -78A > G (-28) carriers had amplification products (bands indicated by arrows in the electrophoresis) at annealing temperatures of 63 and 64 ℃ and no amplification products when the annealing temperature was increased to 65 and 66 ℃, indicating that HBB-28 primers F and R specifically templated and amplified products from HBB c. -78A > G (-28) carrier gDNA at the appropriate annealing temperature (63 or 64 ℃).

The annealing temperatures of the other 2 pairs of PCR amplification primers were searched in the same manner, and the appropriate annealing temperatures for the 3 pairs of primers all contained 63 ℃ so that the annealing temperatures were selected and PCR reaction conditions were established for subsequent detection, as shown in FIG. 6.

(3) 3 parts of each of HBB: C. -78A > G (-28), HBB: c.126_129delCTTT (CD41-42), HBB: c.316-197C > T (IVS-II-654) and normal human gDNA samples (the concentration is 10 ng/. mu.L) are collected for verification of the detection method. The 12 samples were subjected to beta-thalassemia gene detection (PCR + flow-through hybridization) and the mutation sites were defined.

And (3) detecting the mutation sites in a set manner. And (3) taking gDNA of a blank control, a normal person, a-28 mutation carrier, a CD41-42 mutation carrier and an IVS-II-654 mutation carrier for detection. Preparing a PCR mixed primer, wherein the components and the concentration in the mixed PCR primer are as follows:

collective detection PCR primer F

HBB-28 primer F10. mu.M

HBB CD41-42 primer F10. mu.M

HBB IVS-II-654 primer F10. mu.M

Collective detection PCR primer R

HBB-28 primer R10. mu.M

HBB CD41-42 primer R10. mu.M

HBB IVS-II-654 primer R10. mu.M

Performing PCR amplification by using the collective PCR primers, wherein the PCR amplification system comprises the following steps:

the PCR reaction conditions are shown in FIG. 6.

The PCR amplification products are detected by a Cas12a fluorescent probe system, each PCR product is provided with 3 multiple holes, and the Cas12a fluorescent probe reaction system is as follows:

after capping, the cells were briefly centrifuged, incubated at 37 ℃ and the reaction was started to read the initial fluorescence value, after which the fluorescence value was read 1 time every 1 min. Δ fluorescence value — end fluorescence value (30min) — initial fluorescence value (0 min).

② detecting single mutation site. And 3 groups are divided for detection, namely a-28 mutation site group (blank control, normal person and-28 mutation carrier), a CD41-42 mutation site group (blank control, normal person and CD41-42 mutation carrier), and an IVS-II-654 mutation site group (blank control, normal person and IVS-II-654 mutation carrier), wherein each group is amplified by using corresponding PCR primers.

The PCR amplification system is as follows:

the PCR reaction conditions are shown in FIG. 6.

③ detecting result

Firstly, 3 mutation sites are detected through 1 reaction system set, after primers aiming at the 3 mutation sites are mixed, gDNAs of a carrier and a normal person are amplified respectively, and water is used as a negative control. The amplification products were detected using Cas12 a-fluorescent probe system, and the fluorescence values of 9 carriers were higher than those of normal human and negative controls, suggesting that 9 carriers carried at least 1-28, CD41-42 or IVS-II-654 mutations (FIG. 7 a). The method can distinguish normal people from carriers on a plurality of mutation sites, is suitable for detecting whether an individual carries the mutation of the beta-thalassemia, but if specific mutation sites need to be determined after the individual is detected as the carrier, a single site needs to be further detected. Next, mutation site detection was performed on-28, CD41-42 and IVS-II-654 in 9 carriers, and

carriers

1, 2 and 3 were carriers of-28 mutation,

carriers

4, 5 and 6 were carriers of CD41-42 mutation, and

carriers

6, 7, 8 and 9 were carriers of IVS-II-654 mutation, respectively, as judged by fluorescence intensity (FIG. 7 b). The 12 samples had been previously confirmed by clinical examination for genotype, and the results of the collective test and the single mutation site test were consistent with the clinical examination results.

The PCR amplification primers designed and synthesized aiming at mutation sites HBB: c. -78A > G (-28), HBB: c.126-129 delCTTT (CD41-42) and HBB: c.316-197C > T (IVS-II-654) are fixed except for PAM sequence, and the primer sequence recognized by the target gene is designed according to the target gene sequence, but the sequence designed by the application has better specificity and accuracy compared with other recognition primer sequences designed according to the target gene sequence in a database, and is more suitable for judging mutation by adopting the method disclosed by the invention.

Example 2

Comparative experiment method-existing Cas12a detection method and principle detection

HBB c. -78A detection method by using existing Cas12a detection method and principle>G. HBB: c.126-129 delCTTT and HBB: c.316-197C>T, and the like, 3 mutation sites, namely, a PAM sequence needs to be searched near the mutation sites and a proper crRNA needs to be designed. The design was performed using crRNA design software (CRISPR RGEN Tools) and found that: c. 78A of HBB>There was no PAM sequence (TTTN) near the G mutation site, and crRNA could not be designed (FIG. 8 a); c.126-129 delCTTT (Hilbert-Kammlung cancer) mutant site, a PAM sequence (TTTN) is arranged near the mutant site, but the designed crRNA does not cover the mutant site (FIG. 8 b); c.316-197C HBB>There is PAM sequence (TTTN) near the T mutation site and 2 crRNAs can be designed, but the mutation siteAt the middle or 3' end of the crRNA recognition region, which is far from the PAM sequence, 2 crrnas recognize the normal site and the mutation site with poor specificity (fig. 8 c). Since Cas12a has a certain tolerance for base mismatch between crRNA and the target sequence, non-target sequences may also be recognized by Cas12a, affecting the specificity of Cas12a nucleic acid detection, which is also the reason why CRISPR/Cas system may be off-target in gene editing. Kleinstein and Kim et al^[9,10]It was found that Cas12a has tolerance to mismatches related to the number and position of mismatched bases, Cas12a has high tolerance to mismatches and low specificity to discriminate between different bases when the mismatched bases are far from the PAM sequence, and Cas12a has low tolerance to mismatches and high specificity to discriminate between different bases when the number of mismatched bases is large or the distance is close to the PAM sequence.

On 3 mutation sites such as HBB: C. -. 78A > G, HBB: c.126-129 delCTTT and HBB: c.316-197C > T, 3 mutation sites cannot be detected by the existing Cas12a detection method due to no PAM sequence or no proper crRNA design.

The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.

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Claims

1. A detection method based on Cas protein is characterized by comprising the following steps:

s2, carrying out PCR amplification on the target gene by adopting a PCR amplification primer to obtain an amplification product; then, detecting an amplification product by using a Cas protein, crRNA and fluorescent probe system; the sequences of the primers comprise a PAM sequence and a crRNA recognition region sequence.

2. The assay of claim 1, wherein the Cas protein is Cas12a, Cas13, Cas12b, or Cas 14; preferably, the Cas protein is Cas12 a.

3. A PCR amplification primer comprising a PAM sequence and a crRNA recognition region sequence.

4. The PCR amplification primer according to claim 3, wherein the PAM sequence is TTTN (SEQ ID No.1) or TTN (SEQ ID No. 2); preferably, the crRNA recognition region sequence comprises 18-23 bases.

5. The PCR amplification primers of claim 3, wherein the 3' base of one of the amplification primers is a locked nucleic acid modified base.

6. The PCR amplification primer of claim 3, wherein the sequence of the PCR amplification primer is shown as SEQ ID No. 3-8.

7. Use of the PCR amplification primer of claim 6 in the preparation of a reagent for detecting β -thalassemia.

8. A kit comprising the PCR amplification primers of any one of claims 3 to 6; preferably, the kit further comprises a crRNA for identifying the PCR amplification primer according to any one of claims 3 to 6.

9. Use of the kit of claim 8 for the preparation of a reagent for the detection of β -thalassemia.

10. The detection method according to claim 1, wherein the target gene is human β -globin gene (HBB), and its mutation site is HBB: c. -78A > G (-28), HBB: c.126_129delCTTT (CD41-42) or HBB: c.316-197C > T (IVS-II-654).