CN113174433B - Cas protein-based detection method - Google Patents
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Abstract
The invention relates to a detection method based on Cas protein, which comprises the following steps: s1, designing a synthetic PCR amplification primer according to a 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; detecting the amplified product by using a Cas protein, crRNA and fluorescent probe system; the primer sequence comprises a PAM sequence and a crRNA recognition region sequence. The invention breaks through the limitations of PAM sequences and crRNA, and can detect by manually introducing the PAM sequences and crRNA through the primers even if no PAM sequences exist near the mutation site to be detected or proper crRNA cannot be designed. And the primer-mediated Cas12a qualitative detection can detect a plurality of variant sites in 1 reaction system, thus reflecting the capability of multiple detection, being beneficial to reducing operation steps and reducing detection cost.
Description
Technical Field
The present invention relates to a method for detecting nucleic acid molecules based on Cas proteins.
Background
Clustered regularly interspaced short palindromic repeats (clustered regularly interspaced short palindrome repeats, CRISPR) and CRISPR-associated nucleases (CRISPR associated proteins, cas) ubiquitous in bacteria and archaebacteria are the defenses of bacteria against exogenous plasmid or phage infection, and scientists have successively discovered a variety of Cas proteins and their mechanisms of operation. Cas12a (also known as Cpf 1) specifically recognizes and is activated under crRNA guidance, and activated Cas12a can specifically cleave both a target dsDNA molecule and non-specifically cleave an unrelated single-stranded DNA (ssDNA), i.e., an accessory cleavage activity.
Detection of target dsDNA molecules using the incidental cleavage activity of Cas12a, prior art protocol principle: (1) designing a primer according to the sequence of the target dsDNA molecule to be detected, amplifying the target dsDNA molecule by a PCR or isothermal amplification method, if the target dsDNA molecule exists, designing and synthesizing crRNA according to the sequence of the target dsDNA molecule to be detected, leading the crRNA to the Cas12a to recognize the target dsDNA molecule, (3) taking the reaction solution of the amplification system, incubating with the Cas12a, the crRNA and the probe ssDNA (fluorescent probe), and (4) detecting whether the Cas12a is activated or not by an instrument after the incubation is finished, thereby carrying out qualitative detection on the target dsDNA molecule.
Chen et al combine Cas12a with recombinase polymerase isothermal amplification (recombinase polymerase amplification, RPA) technology, li et al combine Cas12a with PCR technology, and respectively develop nucleic acid detection technologies DETECTER and HOLMES based on Cas12a, the basic principle is: the crRNA is designed according to the target dsDNA, amplified target dsDNA molecules can be specifically recognized and cut by the Cas12a-crRNA complex, cas12a is activated at the same time, the ssDNA probe is cut by utilizing the auxiliary cutting activity of the Cas12a after activation, the two ends of the probe are respectively marked with a fluorescent group and a quenching group, the fluorescent signal is released after the probe is cut, and the target dsDNA is detected by reading the detection signal released after the probe is cut (figure 1) [1,2] 。
However, cas12 a-based nucleic acid detection techniques suffer from the following drawbacks:
PAM sequence restriction
The first precondition for Cas12a to recognize target dsDNA is: the target dsDNA must be present with the corresponding PAM sequence TTTN or TTN. Only when PAM sequences are present, it is possible to design crrnas 3' downstream of PAM sequences and detect them (fig. 2). If no PAM sequence is present in the target sequence, then crRNA cannot be designed and therefore cannot be detected with Cas12a. PAM sequences greatly limit the range of choice of target sequences and flexibility in crRNA design.
Crrna restriction
A second precondition for Cas12a to recognize 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 detection specificity. Even if PAM sequence is present in the target sequence to be detected, if crRNA with high specificity cannot be designed downstream of PAM (fig. 2), it may result in a decrease in the specificity of detection and thus cannot be detected. Because Cas12a has some tolerance for base mismatch between crRNA and target sequence, it is also the reason that CRISPR/Cas systems 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 the target nucleic acid molecule exists or not, 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 is often a concentration difference between samples, and in order to compare the copy number difference between samples in the target nucleic acid, an internal reference needs to be set up to eliminate the quantitative detection result difference caused by the sample concentration difference.
4. Limited to pathogen detection
The pathogen genome sequence is relatively large, so that a proper PAM sequence is hopeful to be found and crRNA with higher specificity is designed, and therefore, a plurality of documents report that Cas12a nucleic acid detection technology is applied to Zika virus and dengue virus at present [5] Novel coronavirus pneumovirus [6,7] And Mycobacterium tuberculosis [8] Qualitative detection of a variety of pathogens, etc.
Common types of variation in genetic disease include point mutations, small insertions, or deletions. The small insertions or deletions of point mutations, which are limited to 1 or a few bases, often lack PAM sequences in the vicinity or make it difficult to design a suitable crRNA, and thus are difficult to detect using current Cas12a nucleic acid detection techniques. Whereas beta-thalassemia is mainly caused by point mutations in the gene encoding globin.
Thalassemia (thalassimia) is a disease in which the gene encoding globin is mutated, the synthesis of globin is reduced or completely deleted, and the chain of globin is unbalanced, thereby causing hereditary hemolytic anemia. The carrying rate and the incidence rate of alpha-thalassemia and beta-thalassemia in the population in the south of China are higher. Thalassemia patients clinically manifest as hemolytic anemia, developmental delay, special face and hepatosplenomegaly, whereas the carriers generally have no obvious clinical symptoms or merely manifest as changes in hematological indicators such as reduced mean erythrocyte volume (mean corpuscular volume, MCV), reduced mean erythrocyte hemoglobin content (mean corpuscular hemoglobin, MCH) and abnormal hemoglobin.
Currently, domestic screening of beta-thalassemia carriers mainly adopts a screening strategy based on a hematological phenotype, and MCV, MCH reduction and HbA2 increase are positive indexes of the beta-thalassemia carriers. Molecular detection is performed after screening out the carrier to determine the genotype of the carrier, but the screening strategy may miss carriers whose hematological phenotype is negative, and may misjudge individuals with iron deficiency anemia as carriers.
Since the mutation type of beta-thalassemia is mainly point mutation, the mutation is limited to 1 or a few bases, and PAM sequences are often lacking nearby or it is difficult to design proper crrnas, so that detection by the existing Cas12a nucleic acid detection technology is difficult.
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 PAM sequences and crRNA recognition regions through a primer-mediated strategy, and the PAM sequences and the crRNA recognition regions on target dsDNA are not relied on any more, so that the target dsDNA can be detected even if the target dsDNA has no PAM sequences 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 a synthetic PCR amplification primer according to a 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; detecting the amplified product by using a Cas protein, crRNA and fluorescent probe system;
the primer sequence comprises a PAM sequence and a crRNA recognition region sequence.
Preferably, the Cas protein is Cas12a, cas13, cas12b or Cas14.
Further preferred, the Cas protein is Cas12a.
Cas12a and Cas12b can directly recognize double-stranded DNA, 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 example is double-stranded DNA, and can be directly recognized by Cas12a or Cas12 b. If Cas13 is used, detection steps are added to transcribe single stranded RNA molecules from the target double stranded DNA molecules before detection can be performed. If Cas14 is used, additional detection steps are required to degrade 1 strand of the target double-stranded DNA molecule to form a single-stranded DNA molecule before detection can be performed.
The invention claims a PCR amplification primer, which comprises PAM sequence and 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 3' end base of one of the amplification primers is a locked nucleic acid modified base.
The lock nucleic acid modification can not only improve the annealing temperature, but also improve the specificity.
Taking the HBB-28 mutation site as an example, one of the amplification primer species, such as F, has a sequence designed based on the sequence of the HBB: c. -78A > G mutation site, which is a normal base, and G after mutation. In order to ensure the specificity of detection, the amplification primer F needs to specifically recognize the sequence after mutation in the PCR process and combine with the sequence to amplify the product, but can not combine with the normal sequence and amplify the product. Because F has no mismatch with the mutant sequence and F has 1 mismatch with the normal sequence, the annealing temperature of F with the mutant sequence is higher than that of F with the normal sequence. Theoretically, the difference in annealing temperatures can be used to amplify the mutant sequences at higher annealing temperatures, but not the normal sequences. In order to further increase the annealing temperature and the specificity of amplified mutation sites, the 3' -terminal base of F is modified by a locked nucleic acid, and the annealing temperature of the primer after the locked nucleic acid modification is increased by 3-8 ℃. Higher annealing temperatures can be set during PCR to specifically amplify sequences containing the mutation sites, but not 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 comprising the PCR amplification primer.
The invention claims the application of the kit in preparing the reagent for detecting beta-thalassemia.
The present invention claims a crRNA for use in identifying PCR amplification primers.
The invention claims the application of the crRNA in preparing the reagent for detecting beta-thalassemia.
The invention claims a kit comprising the crRNA described above.
The invention claims the application of the kit in preparing the reagent for detecting beta-thalassemia.
The invention claims a kit comprising the above-described PCR amplification primers, and crRNA for identifying the PCR amplification primers.
The invention claims the application of the kit in preparing the reagent for detecting beta-thalassemia.
Preferably, the target gene is a human beta-globin gene (HBB) gene and a mutation site HBB: c-78A > G (-28), HBB: c.126_129delCTTT (CD 41-42) or HBB: c.316-197C > T (IVS-II-654).
The PCR amplification primers are shown in table 1:
TABLE 1 primers, probes and crRNA sequence information
The invention claims a kit comprising PCR amplification primers shown as SEQ ID NO.3-8 and crRNA shown as SEQ ID NO. 10.
The invention claims the application of the kit in preparing the reagent for detecting beta-thalassemia.
The use may be for non-disease diagnostic purposes.
The incidental cleavage activity of the Cas12a cleavage probe is nonspecific, and the probe sequence is a single-stranded DNA, so that no special requirement exists.
Preferably, the PCR amplification reaction system is as follows:
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 detected simultaneously in one reaction system, and multiple detection is realized. After the subject is subjected to collective detection (3 mutation sites are detected simultaneously), it can be determined whether the subject carries 3 mutation sites. Technical advantages are that: (1) multiple detection, 1 detection can screen 3 mutation sites, so that the cost-benefit of detection is improved; (2) and a plurality of body change sites are detected in a collecting way, so that the method is suitable for the requirement of population screening.
Preferably, the PCR reaction conditions are as 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 and carriers on a plurality of mutation sites, is suitable for detecting whether individuals carry the mutation of beta-thalassemia, and if the individuals need to determine specific mutation sites after the individuals are detected as carriers, the individual sites need to be further detected.
Preferably, the mutation site detection is performed in 3 sets, each set being amplified with a corresponding PCR primer.
Preferably, the PCR amplification system is as follows:
preferably, the PCR reaction conditions are as shown in FIG. 6.
The invention is further explained below:
the 3 mutation sites can not be detected by the existing Cas12a nucleic acid detection technology by taking 3 common mutations of beta-thalassemia HBB: c-78A > G (-28), HBB: c.126-129 delCTTT (CD 41-42) and HBB: c.316-197C > T (IVS-II-654) in Chinese population as application examples. The crRNA can not be designed by utilizing crRNA on-line design software, and the crRNA can not be designed by utilizing the mutation site of HBB: c. -78A > G, and the crRNA can be designed by utilizing the mutation site of HBB: c.126-129 delCTTT and HBB: c.316-197C > T, but the specificity of the designed crRNA is poor, the off-target effect exists, and the detection can not be carried out.
Taking the mutation of HBB: c. -78A > G (-28) as an example, normal people have no mutation, the 3' -end base of the HBB-28 primer F is G modified by a locked nucleic acid, the locked nucleic acid cannot be completely complementary with the HBB gene, the locked nucleic acid modification can raise the annealing temperature, inhibit the HBB-28 primer F from annealing with the wild type HBB gene to form double chains, and theoretically, the product cannot be amplified. The HBB-28 primer F/R is completely complementary with the mutated HBB gene by the mutation carrier-28, and the product can be amplified by PCR. The amplified product is dsDNA, and contains PAM sequence and universal crRNA recognition region sequence in addition to the corresponding sequence of HBB gene, and is complementary to 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 the fluorescent signal. Whether the site was mutated or not was 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:
(1) the limitations of PAM sequences and crRNA are broken through, and even if no PAM sequence exists near the mutation site to be detected or a proper crRNA can not be designed, the PAM sequences and crRNA can be artificially introduced through the primers for detection.
(2) The primer-mediated Cas12a qualitative detection can detect a plurality of variant sites in 1 reaction system, thus reflecting the capability of multiple detection, being beneficial to reducing operation steps and reducing detection cost.
Drawings
FIG. 1 is a schematic diagram of a nucleic acid detection technique based on Cas12a in the prior art;
FIG. 2 is a schematic representation of PAM sequences and crRNA recognition regions;
FIG. 3 is a schematic diagram of the primer-mediated Cas12 a-based qualitative detection principle of the present invention;
FIG. 4 is a PCR reaction condition of 4 annealing temperatures that was sought by the present invention;
FIG. 5 is an experimental result of the invention for exploring a suitable annealing temperature;
FIG. 6 shows the PCR conditions established in the present invention
FIG. 7 is a primer-mediated qualitative detection of Cas12a by normal humans and beta-thalassemia carriers of the present invention;
fig. 8 is a schematic diagram of PAM sequence and crRNA design for detecting the vicinity of 3 mutation sites of HBB gene using the existing Cas12a detection method.
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Example 1
(1) According to the human HBB gene sequence, 3 common mutation sites HBB: c. -78A > G (-28), HBB: c.126_129delCTTT (CD 41-42) and HBB: c.316-197C > T (IVS-II-654) of Chinese people are selected, and PCR amplification primers are designed and synthesized as shown in Table 1.
TABLE 1 primers, probes and crRNA sequence information
(2) The primer was verified and the annealing temperature was fuelled.
(1) Taking HBB-28 primer F and R as examples, 1 part of negative control (water), HBB: c. -78A > G (-28) carrier and normal human gDNA sample are selected, and 4 PCR amplification systems are respectively prepared for each sample.
The PCR amplification system was as follows:
(2) the PCR reaction conditions were substantially as shown in FIG. 4, except that the annealing temperatures were set to 63, 64, 65 and 66℃respectively, the annealing times were all 30s, and the other conditions were the same. Each sample was amplified under 4 different reaction conditions, and the amplified products were subjected to agarose gel electrophoresis.
(3) As shown in FIG. 5, the electrophoresis results show that the amplified products of the negative control (water) and normal people under 4 annealing temperature conditions have no obvious bands after electrophoresis, which indicates that the primer specificity is better and the non-specific products are not amplified. HBB.c. -78A > G (-28) carriers had amplified products (bands indicated by arrows in the electrophoreses) at annealing temperatures of 63 and 64℃and no amplified products when the annealing temperatures were raised to 65 and 66℃indicating that HBB-28 primers F and R were able to specifically template HBB.c. -78A > G (-28) carrier gDNA and amplify the products at the appropriate annealing temperatures (63 or 64 ℃).
The annealing temperatures of the other 2 pairs of PCR amplified primers were searched for in the same way, 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 the subsequent detection as shown in FIG. 6.
(3) 3 samples of gDNA from HBB: c. -78A > G (-28), HBB: c.126-129 delCTTT (CD 41-42), HBB: c.316-197C > T (IVS-II-654) were collected (at a concentration of 10 ng/. Mu.L) each for validation of the assay. All 12 samples were tested for beta-thalassemia gene (PCR+flow-directed hybridization) and mutation sites were defined.
(1) 3 mutation site sets were detected. Blank, normal, -28 mutant carrier, CD41-42 mutant carrier and IVS-II-654 mutant carrier gDNA were taken for detection. Preparing a PCR mixed primer, wherein the components and the concentrations of the mixed PCR primer are as follows:
PCR primer F for collection detection
HBB-28 primer F10. Mu.M
HBB CD41-42 primer F10. Mu.M
HBB IVS-II-654 primer F10. Mu.M
PCR primer R for collection detection
HBB-28 primer R10. Mu.M
HBB CD41-42 primer R10. Mu.M
HBB IVS-II-654 primer R10. Mu.M
PCR amplification was performed using pooled PCR primers, the PCR amplification system was as follows:
the PCR reaction conditions are shown in FIG. 6.
The PCR amplified products are detected by a Cas12a fluorescent probe system, 3 compound holes are arranged in each PCR product, and the Cas12a fluorescent probe reaction system is as follows:
after capping, centrifugation was performed briefly, incubation was performed at 37℃and the reaction was started to read the initial fluorescence value, after which 1 fluorescence value was read every 1 min. Delta fluorescence value = endpoint fluorescence value (30 min) -initial fluorescence value (0 min).
(2) Single mutation site detection. The detection was performed in 3 groups, -28 mutant site groups (blank, normal, -28 mutant carrier), CD41-42 mutant site groups (blank, normal, CD41-42 mutant carrier), IVS-II-654 mutant site groups (blank, normal, IVS-II-654 mutant carrier), each group was amplified with the corresponding PCR primer.
The PCR amplification system was as follows:
the PCR reaction conditions are shown in FIG. 6.
The PCR amplified products are detected by a Cas12a fluorescent probe system, 3 compound holes are arranged in each PCR product, and the Cas12a fluorescent probe reaction system is as follows:
after capping, centrifugation was performed briefly, incubation was performed at 37℃and the reaction was started to read the initial fluorescence value, after which 1 fluorescence value was read every 1 min. Delta fluorescence value = endpoint fluorescence value (30 min) -initial fluorescence value (0 min).
(3) Detection result
First, 3 mutation sites were detected by 1 reaction system set, and primers for 3 mutation sites were mixed to amplify gDNA of the carrier and normal person, respectively, using water as a negative control. The amplified products were detected with Cas12 a-fluorescent probe system, with fluorescence values higher for 9 carriers than for normal human and negative control, 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 and carriers on a plurality of mutation sites, is suitable for detecting whether individuals carry the mutation of beta-thalassemia or not, but if the specific mutation sites are required to be clear after the detection of the individuals as the carriers, the single sites are required to be further detected. Next, mutation site detection was performed on the-28, CD41-42 and IVS-II-654 cases for 9 cases, respectively, and it was determined that the carriers 1, 2 and 3 were the-28 mutation carriers, the carriers 4, 5 and 6 were the CD41-42 mutation carriers, and the carriers 6, 7, 8 and 9 were the IVS-II-654 mutation carriers according to the fluorescence intensity (FIG. 7 b). The genotype of 12 samples was previously clarified by clinical testing, whereas the results of the pooled testing and single mutation site testing were consistent with the clinical testing results.
The PCR amplification primers designed and synthesized aiming at mutation sites HBB: c-78A > G (-28), HBB: c.126-129 delCTTT (CD 41-42) and HBB: c.316-197C > T (IVS-II-654) are fixed except for the PAM sequence, and the primer sequence identified with the target gene is designed according to the target gene sequence, but the designed sequence has better specificity and accuracy compared with other identification primer sequences designed according to the target gene sequence in a database, and is also more suitable for judging mutation by adopting the method.
Example 2
Comparative experiment method-existing Cas12a detection method and principle detection
HBB: c. -78A is detected by applying the existing Cas12a detection method and principle>G. HBB, c.126-129 delCTTT and HBB, c.316-197C>T and other 3 mutation sites, and first, a PAM sequence needs to be searched near the mutation sites and a proper crRNA is designed. Design using crRNA design software (CRISPR RGEN Tools), found: (1) HBB c. -78A>No PAM sequence (TTTN) was present near the G mutation site and crRNA could not be designed (fig. 8 a); (2) HBB, wherein a PAM sequence (TTTN) is arranged near the c.126-129 delCTTT mutation site, but the designed crRNA does not cover the mutation site (FIG. 8 b); (3) HBB c.316-197C>A PAM sequence (TTTN) is arranged near the T mutation site, 2 crRNAs can be designed, but the mutation site is positioned at the middle part or the 3' -end of the crRNA recognition region and is far away from the PAM sequence, and the specificity of 2 crRNAs for recognizing the normal site and the mutation site is poor (figure 8 c). Because Cas12a has a certain tolerance to base mismatch between crRNA and target sequence, non-target sequences may also be recognized by Cas12a, affecting the specificity of Cas12a nucleic acid detection, which is also why CRISPR/Cas systems may be off-target in gene editing. Kleinsiver and Kim et al [9,10] The tolerance of Cas12a to mismatches was found to be related to the number and position of mismatched bases, with Cas12a being tolerant to mismatches when the mismatched bases are far from the PAM sequenceHigh specificity to distinguish between different bases, while Cas12a has low tolerance to mismatches when the number of mismatched bases is large or is close to PAM sequence, the specificity to distinguish between different bases is high.
On 3 mutation sites such as HBB: c-78A > G, HBB: c.126-129 delCTTT, and HBB: c.316-197C > T, the existing detection method of Cas12a cannot detect the 3 mutation sites because no PAM sequence or proper crRNA cannot be designed.
The foregoing examples are set forth in order to provide a more thorough description of the present invention, and are not intended to limit the scope of the invention, since modifications of the invention in various equivalent forms will occur to those skilled in the art upon reading the present invention, and are within the scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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caccatggtg tctgtttgag 20
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tgctctgtct gcatgggttt ggcacaatga agtgggtaac ctttcagtgg acccagaggt 60
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Claims (7)
1. A Cas protein-based detection method for non-disease diagnosis or treatment purposes, comprising the steps of:
s1, designing a synthetic PCR amplification primer according to a 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; detecting the amplified product by using a Cas protein, crRNA and fluorescent probe system; the sequence of the primer comprises a PAM sequence and a crRNA recognition region sequence; the sequence of the PCR amplification primer is shown as SEQ ID NO. 3-8; the 3' -end base of the upstream primer in the PCR amplification primer is a base modified by the locked nucleic acid; the sequence of the crRNA is shown as SEQ ID NO. 10;
the Cas protein is Cas12a;
the target gene is a human beta-globin gene, and comprises mutation sites HBB: c. -78A > G, HBB: c.126-129 delCTTT or HBB: c.316-197C > T.
2. A PCR amplification primer, wherein the PCR amplification primer comprises a PAM sequence and a crRNA recognition region sequence; the 3' -end base of the upstream primer in the amplification primer is a base modified by the locked nucleic acid;
the sequence of the PCR amplification primer is shown as SEQ ID NO. 3-8.
3. The PCR amplification primer of claim 2, wherein the PAM sequence is TTTN (SEQ ID No. 1) or TTN (SEQ ID No. 2); the crRNA recognition region sequence comprises 18-23 bases.
4. The use of a PCR amplification primer as claimed in claim 2 in the preparation of a reagent for detecting β -thalassemia, which is a reagent for detecting the human β -globin gene HBB: c, -78a > g, HBB: c.126_129delCTTT or HBB: c.316-197c > t.
5. A kit comprising the PCR amplification primer of any one of claims 2-3.
6. The kit of claim 5, further comprising crRNA for recognizing the PCR amplification primers of any one of claims 2 to 3, wherein the crRNA has a sequence as shown in SEQ ID No. 10.
7. The use of the kit according to claim 5 for the preparation of a reagent for detecting beta-thalassemia, which is a reagent for detecting human beta-globin gene HBB: c. -78A > G, HBB: c.126_129delCTTT or HBB: c.316-197C > T.
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