CN116179511A - Application of Cpf1 protein in preparation of kit for nucleic acid detection - Google Patents
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Abstract
The invention discloses an application of Cpf1 protein in preparing a kit for nucleic acid detection, and relates to the field of biotechnology. When the novel nuclease is used for detecting the nucleic acid fragments, the defects of the traditional Cas12a can be overcome, the identification range of PAM is increased, the reaction speed is accelerated, the signal intensity during detection is obviously improved, the detection time is shortened, the detection efficiency is improved, the sensitivity is high, the specificity is high, the accuracy is high, the detection is visual (the visual detection can be directly carried out by naked eyes under a fluorescent lamp), the cost is low, complex large-scale experimental equipment is not needed, and the operation is simple and convenient. These advantages make the detection method of the present invention more suitable for rapid detection and identification diagnosis of primary experiments and clinical lines.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of Cpf1 protein in preparation of a kit for nucleic acid detection.
Background
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats, CRISPRs) system originally derived from the RNA-guided adaptive immune system of bacteria for protection against the nucleic acid components of invading bacteria and archaea, CRISPR refers to clustered, regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats), derived from phage DNA fragments that can infect prokaryotes, which can detect and destroy similar DNA in other phages that can cause similar infection, and thus are critical for prokaryote anti-phage, so this sequence is the immune system of many prokaryotes. Cas protein refers to CRISPR-associated protein, which is a related protein in a CRISPR system. The CRISPR locus consists of an operon encoding a Cas protein and a repeat spacer, and can be used to recognize and cleave a DNA strand specific for its complement using RNA guidance corresponding to the spacer (spacer) in the CRISPR sequence, i.e., cas protein targeted degradation of foreign nucleic acids by RNA-guided nucleases ([ 1] r, b., et al, CRISPR provides acquired resistance against viruses in prokaryotes. Science (New York, n.y.), 2007, 315 (5819): p.1709-12 @ [2]LA,M.and S.EJ,CRISPR interference limits horizontal gene transfer in staphylococci by targeting DNA.Science (New York, n.y.), 2008, 322 (5909): p.1843-5.). Wherein CRISPR-Cpf1 (Cpf 1) belongs to the Cas enzyme second family, used to guide RNA-guided double-stranded DNA cleavage of a single RuvC catalytic domain. Cpf1 enzymes recognize The Thymine (T) nucleotide rich spacer adjacent motif (PAM) (B, Z., et al, cpf1 is a single RNA-guided endonuclease of a class 2CRISPR-Cas system. Cell,2015, 163 (3): p.759-71. The term explains PAM refers to The proscenium sequence adjacent motif (protospacer-adjacent motif) necessary for cleavage by Cas12, the PAM of FnCas12a is The TTN sequence, the PAM of LbCAs12a is The TTTV (V reference A, C or G) sequence), catalyzes their own guided CRISPR RNA (crRNA) maturation (I, F., et al, the CRISPR-associated DNA-cleaving enzyme Cpf1 also processes precursor CRISPR RNA. Nature,2016, 532 (7600): p.517-21), and specifically recognizes and cleaves complementary paired double-stranded DNA (B, Z., et al, cpf1 is a guided endonuclease of a class-163.71, and CRISPR-163.75. When the CRISPR/Cpf1 protein recognizes the cleavage target double-stranded DNA in a sequence-specific manner, a powerful non-specific single-stranded DNA (ssDNA) trans-cleavage activity (JS, c., et al, CRISPR-Cpf1 target binding unleashes indiscriminate single-structured DNase activity.science (New York, n.y.), 2018, 360 (6387): p.436-439) can be induced.
Cpf1 is an endonuclease active ligand ion, typically magnesium. The existing Cpf 1-based nucleic acid detection has the defects that the signal intensity is still not high, so that the detection time in the actual detection is too long, the detection efficiency of a low-concentration sample is low, and the like.
The CRISPR-Cas system provides powerful tools for gene regulation and genome engineering, and has great application potential in the field of gene editing and molecular diagnosis. In particular, based on the parachuting activity of Cpf1, diagnostic tools for detecting nucleic acids with high specificity can be developed, and even Single Nucleotide Polymorphisms (SNPs) can be distinguished. Under crRNA mediation, the Cpf1 protein binds to the target dsDNA sequence, activating cis-cleavage activity while simultaneously exhibiting non-specific parachuting activity on non-specific target ssDNA. Based on the parachuting activity of Cpf1, it is particularly suitable for developing dsDNA target sequence detection for detecting viral or genomic mutation sites. Although Cpf1 is already available for specific detection based on the present day, there is still a further perfection. The first challenge of Cpf1 protein detection is that Cpf1 protein activity is limited by the proximity motif of the pro-region sequence and that only a partial nucleic acid target, such as LbCPf1, is likely to recognize a TTTV-containing PAM sequence. The second challenge of Cpf1 protein detection is the limited speed of the reaction, and it is difficult to reach saturation signal rapidly in a short time. The activity of traditional Cas12 nucleases such as increased detection sensitivity, and the development of novel Cas12a proteases with broader PAM recognition range.
Disclosure of Invention
The invention aims to solve the technical problems of Cas12a detection dsD in the prior artThe NA target sequence has the defects of limited PAM range, insufficient detection sensitivity, long detection time and the like. Provides an application of novel Cas12a nuclease in preparing a kit for nucleic acid detection, mn is added 2+ The ions can activate their bypass cleavage activity, thereby achieving the detection goal. The PAM recognition range of the nuclease is larger than that of the traditional nuclease, the reaction speed is faster, and the detection is faster and more sensitive.
The specific technical scheme is as follows:
the invention provides application of Cpf1 protein in preparing a kit for detecting nucleic acid, wherein the amino acid sequence of the Cpf1 protein is shown as SEQ ID NO. 1.
The invention also provides a nucleic acid detection kit comprising a Cpf1 detection system, the Cpf1 detection system comprising: guide RNA, cpf1 protein, nucleic acid probe and manganese ion-containing solution;
the guide RNA is crRNA for guiding Cpf1 protein to specifically bind to the nucleic acid;
the amino acid sequence of the Cpf1 protein is shown as SEQ ID NO. 1.
Preferably, the solution containing manganese ions may be manganese sulfate, manganese chloride or manganese acetate solution. In the example of the embodiment of the present invention, it is preferable that the solution containing manganese ions is a manganese chloride solution;
and/or, the nucleic acid probe is a single-stranded DNA probe.
Preferably, the nucleic acid probe is a single-stranded DNA probe, which preferably includes a fluorescent label, more preferably has a fluorescent group and a fluorescence quenching group at both ends thereof, and even more preferably has a fluorescent group at the 5 'end and a fluorescence quenching group at the 3' end. The fluorophore may be conventional in the art, for example, 6-FAM, TET, CY3, CY5 or ROX.
The fluorescence quenching group may be conventional in the art, for example, BHQ1, BHQ2, or BHQ3. The single-stranded DNA probe may be prepared by a method conventional in the art, and the sequence thereof may be TTATT, for example.
Cas12a can recognize DNA nucleic acid sequences under the guidance of RNA, but traditional Cas12a protein PAM recognitionOther ranges are limited, such as LbCPf1 PAM is TTTV, and the novel Cas12a protease found by us has wider range of recognition PAM and expands the range of recognition. Furthermore, we found that Cas12a protease is found in Mn 2+ In the presence, the cleavage efficiency is greatly improved, and the target molecules can be detected more quickly.
Preferably, in the examples of the embodiments of the present invention, the final concentration of manganese ions in the manganese ion-containing solution used is 10mM.
Specifically, the Cpf1 detection system comprises the following specific components: 200ng Cpf1 protein, 25pM nucleic acid probe, 1. Mu.M crRNA, 2. Mu.L of sample to be tested, and a final volume of 20. Mu.L.
In the invention, once the target nucleic acid of the sample to be detected, crRNA and Cas12a protein form a ternary complex, the complex can cleave other single-stranded DNA molecules in the system. Targeting a target nucleic acid by designing crrnas; adding crRNA and Cas12a protein to a detection system; when the target DNA is present, cas12a forms a ternary complex with crRNA and target DNA, while the complex exerts its trans-cleavage activity and cleaves single-stranded DNA labeled with fluorescent signal (luminescent groups and quenching groups are attached to both ends, respectively, and the luminescent groups can emit light after being cut off), thereby emitting fluorescence. Therefore, by detecting fluorescence, whether the system to be detected contains the target DNA molecule can be known, and the detection principle can be seen in FIG. 1.
Preferably, the detection kit further comprises a reaction buffer solution, wherein the reaction buffer solution comprises NaCl, tris-HCl and BSA (bovine serum albumin); the pH of the reaction buffer was 7.5.
In an example of an embodiment of the invention, the reaction buffer is 100mM NaCl,50mM Tris-HCl, 100. Mu.g/mL BSA, pH 7.5.
In order to solve the technical problems, the invention also provides a method for detecting nucleic acid of non-diagnostic purpose, which utilizes the Cpf1 detection system in the nucleic acid detection kit to detect the nucleic acid.
Preferably, the reaction temperature of the Cpf1 assay system is 37℃and the reaction time of the Cpf1 assay system is 5 minutes.
In the detection method, a nucleic acid quick release reagent is utilized to release nucleic acid in a sample to be detected;
the nucleic acid is obtained by amplification, preferably by RT-RPA amplification of the nucleic acid in the sample to be tested.
The detection method further comprises the step of reading the result, and the result is read by naked eyes under an enzyme-labeled instrument or a fluorescent lamp.
The nucleic acid detection kit for rapidly detecting nucleic acid provided by the invention can be used for fluorescent detection by using an enzyme-labeled instrument or can be used for macroscopic detection by using fluorescence. In the invention, when the nucleic acid probe (for example ssDNA FQ reporter) is used for fluorescence detection by an enzyme-labeled instrument, the detection excitation light is set to 485nm-520nm; when the detection is directly carried out by naked eyes, a light emitter capable of generating a 485nm wavelength light source is selected for detection.
In the present invention, the clinical sample may be usually subjected to an inactivation treatment to release the nucleic acid in the sample to be examined. Under isothermal conditions, the viral RNA is Reverse Transcribed (RT) to obtain cDNA and polymerase amplification (PCR) is performed. The amplification product can bind to Cas12a-crRNA complex, activating trans-cleavage of ssDNA. ssDNA FQ reporter conjugated to ssDNA, labeled with a fluorescent group and a quencher group, produces a fluorescent signal upon cleavage.
The detection method comprises the following specific steps:
(1) Acquiring DNA of a sample to be detected;
(2) Detecting by using a detection system, wherein the detection system reacts for 5 minutes at 37 ℃; the detection system comprises the following specific components: 200ng Cpf1 protein, 25pM nucleic acid probe, 1. Mu.M crRNA,10ng DNA of the sample to be tested, and a final volume of 20. Mu.L; reaction buffer 100mM NaCl,50mM Tris-HCl, 100. Mu.g/mL BSA,10mM MnCl 2 ，pH 7.5；
(3) And (3) detecting the fluorescence value in the detection system in the step (2) by using an enzyme-labeled instrument.
In the present invention, the nucleic acid probe may also be generally referred to as a fluorescence-quenched single-stranded DNA (ssDNA) reporter system, which generally contains a fluorescent group and a quenching group, fluorescence being quenched by the quenching group in an intact state, and then being cleaved during the reaction, thereby emitting fluorescence.
In an example of an embodiment of the invention, the nucleic acid probe is 5 '-end-6-FAM/TTATT/BHQ 1-3' -end.
Preferably, the Cpf1 protein is a codon optimized Cpf1 protein, a prokaryotic codon optimization is carried out on the Cpf1 protein nucleic acid sequences from various sources to obtain sequences, the sequences are constructed into a pET28a expression vector, low-temperature induced soluble protein expression is carried out, and the target protein is obtained through affinity purification and molecular sieve purification.
Term interpretation:
the term crRNA refers to CRISPR RNA, which is a short RNA that directs Cas12a (Cpf 1) to bind to a target DNA sequence.
The term CRISPR refers to clustered, regularly interspaced short palindromic repeats (clustered regularly interspaced short palindromic repeats), which are the immune system of many prokaryotes.
The term Cas protein refers to a CRISPR-associated protein, which is a related protein in a CRISPR system.
The term Cpf1 (also known as Cas12 a) refers to a crRNA-dependent endonuclease, which is an enzyme of type V (type V) in the CRISPR system classification.
The term PAM refers to a protospacer-adjacent motif (protospacer-adjacent motif) that is necessary for Cas12a cleavage, with PAM for FnCas12a being the TTN sequence and PAM for LbCas12a being the TTTN sequence.
The term RT-RPA (reverse transcription-recombinase polymerase amplification) is used in its conventional sense in the art and generally refers to reverse transcription isothermal amplification, also known as reverse transcription recombinase polymerase amplification.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the beneficial effects that:
when the novel nuclease is used for detecting the nucleic acid fragments, the defects of the traditional Cpf1 can be overcome, the identification range of PAM is improved, the reaction speed is accelerated, the signal intensity during detection is obviously improved, the detection time is shortened, the detection efficiency is improved, the sensitivity is high, the specificity is high, the accuracy is high, the detection is visual (the visual detection can be directly carried out by naked eyes under a fluorescent lamp), the cost is low, complex large-scale experimental equipment is not needed, and the operation is simple and convenient. These advantages make the detection method of the present invention more suitable for rapid detection and identification diagnosis of primary experiments and clinical lines.
Drawings
FIG. 1 is a schematic diagram of the detection of a programmable nuclease.
FIG. 2 is a diagram showing expression and purification of nucleases.
FIG. 3 is a diagram of LbCAs12a and Cas12a-68 pair gene fragment (MERS-upE) detection.
Fig. 4 is a graph of the detection of the paraclinic activity of Cas12a proteins by different ions.
Fig. 5 is a graph of efficiency of Cas12-68 cleavage of nucleic acids containing different PAM sequences.
FIG. 6 is a diagram showing detection of Kras gene and its mutant fragment by Cas 12-68.
FIG. 7 is a diagram of detection of nucleic acid fragments in a cell sample by Cas12 a-68.
FIG. 8 is a single base differential fragment detection diagram of LbCPf1 and Cas12a-68 DNA.
Detailed Description
The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
In the present invention, conventional reagents such as Tris-Base (i.e., tris Base, chinese name also referred to as Tris (hydroxymethyl) aminomethane), bovine Serum Albumin (BSA), naCl, tris-HCl (i.e., tris hydrochloride, chinese name: tris (hydroxymethyl) aminomethane hydrochloride), mgSO 4 、MnCl 2 Glycerol and the like are purchased from national drug company; the detection nucleic acid fragment and RNA synthesis were performed by Beijing qingke biotechnology Co., ltd; fluorescenceThe photogroup-labeled ssDNA was synthesized by su Jin Weizhi biotechnology limited.
The detection principle of the invention is shown in fig. 1, and once the target nucleic acid, crRNA and Cas12a protein of the sample to be detected form a ternary complex, the complex can cleave other single-stranded DNA molecules in the system. The method comprises the following steps: targeting a target nucleic acid by designing crrnas; adding crRNA and Cas12a protein to a detection system; when the target DNA is present, cas12a forms a ternary complex with crRNA and target DNA, while the complex exerts its trans-cleavage activity and cleaves single-stranded DNA labeled with fluorescent signal (luminescent groups and quenching groups are attached to both ends, respectively, and the luminescent groups can emit light after being cut off), thereby emitting fluorescence. Therefore, whether the system to be detected contains the target DNA molecule can be known by detecting fluorescence.
The invention also carries out normalization treatment on the detected fluorescence value. The normalization method has two forms, one is to change the number to a fraction between (0, 1) and one is to change the dimensionality expression to a dimensionless expression. In the example of the embodiment of the present invention, a normalization method of changing the number to a decimal between (0, 1) is preferable.
The single-stranded DNA with fluorescent signal label, i.e., single-stranded DNA reporter gene (ssDNA-FQ), has a fluorescent group at the 5 'end and a fluorescence quenching group at the 3' end. The fluorescent group may be 6-FAM, TET, CY3, CY5 or ROX, but is not limited thereto; the fluorescence quenching group may be BHQ1, BHQ2 or BHQ3, but is not limited thereto. The single-stranded DNA is characterized in that two ends of the single-stranded DNA are respectively connected with a luminescent group and a quenching group, and the luminescent groups can emit light after being cut off, so that fluorescence is emitted.
And determining the specificity of the Cas protein for recognizing the PAM sequence by detecting the fluorescence intensity of the reaction system.
The invention will be further illustrated with reference to specific examples.
Example 1: cas12a protease detection target DNA sequence
Preparation of Cas12a protease
In this example, the CPf1 (Lachnospiraceae Cpf, lbCPf 1), also known as LbCAs12a (gene sequence shown as SEQ ID NO. 4), and the novel Cas12a protease gene (gene sequence shown as SEQ ID NO. 2) of the family Maospiraceae are codon optimized to make them more suitable for expression in E.coli strains. The gene was amplified by PCR with NcoI and BamHI primers, and the pET28a plasmid vector was digested simultaneously with NcoI and BamHI, and then the PCR product and plasmid digested products were subjected to cleavage site ligation reaction with T4 ligase. The enzyme-linked reaction products transform DH5 alpha competent cells, and monoclonal is selected for sequencing and identification. Plasmid vectors of the normal insert gene fragments were extracted and BL21 competent cells were transformed. LbCPf1 and Cas12a-68 proteins were expressed in E.coli, bacteria in the medium were collected, sonicated, and purified by nickel column affinity chromatography, eluting the protein of interest using different concentrations of imidazole, wherein the Cas12a-68 protein expression results are shown in FIG. 2, and distinct protein bands were seen in 250 and 500mM imidazole eluates. The amino acid sequences of Cas12a-68 and LbCAs12a used in the experiment are shown as SEQ ID NO.1 and SEQ ID NO. 3.
As shown in FIG. 2, the purified protein is detected by SDS-PAGE, and the purity of the novel nuclease Cas12a-68 can reach more than 85% after being expressed by escherichia coli and purified by a nickel column.
Use of Cas12a protease for detection of MERS virus fragment (MERS-upE)
a. Preparation of nucleic acids
crRNAs were synthesized by beijing engine biotechnology limited, and the sequences of the synthesized crRNAs are shown in table 1.
TABLE 1 crRNA sequence
| crRNA | Sequence (5 '-3') |
| Mers-cr2+ | UaaUUUcUacUaagUgUagaUGACAUAUGGAAAACGAACUAUGU |
| kRas-crRNA1 | UaaUUUcUacUaagUgUagaUagcUUgUggcgUaggcaagagUg |
| kRas-crRNA2 | UaaUUUcUacUaagUgUagaUcUacgccacaagcUccaacUacc |
| kRas-crRNA3 | UaaUUUcUacUaagUgUagaUgcUUgUggcgUaggcaagagUgc |
| kRas-crRNA4 | UaaUUUcUacUaagUgUagaUccacAagcUccaacUaccacaag |
| kRas-crRNA5 | UaaUUUcUacUaagUgUagaUgagcUUgUggcgUaggcaagagU |
| kRas-crRNA6 | UaaUUUcUacUaagUgUagaUccUacgccacAagcUccaacUac |
| kRas-crRNA7 | UaaUUUcUacUaagUgUagaUacgccacAagcUccaacUaccac |
| Let7a-crRNA | UAAUUUCUACUAAGUGUAGAUAACUAUACAACCUACUACCUCA |
CRISPR/Cas12a-Based fluorescence detection
The crRNA in the experiment consisted of a 21nt region that interacted with Cas12a and a 23nt programmable guide region (called spacer) for target DNA recognition, the crRNA sequence is shown in table 1. A single stranded DNA reporter (ssDNA-FQ) labeled with FAM and BHQ1 was synthesized by Suzhou Jin Weizhi Biotechnology Inc., and was 5'-FAM-TTATT-BHQ1-3'.
The detection system is 20 mu L, and the specific components are as follows: 200ng of purified Cas12a (Cas 12a-68 or LbCAs12 a), 25pM ssDNA-FQ, 1. Mu.M crRNA,10ng of dsDNA sample (MERS virus gene fragment, sequence shown in SEQ ID NO. 16) and a final volume of 20. Mu.L. The reaction was incubated at 37 ℃.
In the detection system, crRNA and Cas12a are combined to form ribonucleoprotein complexes, after the Cas12a-crRNA recognizes a target sequence, nuclease activity of the Cas12a is activated, double-stranded DNA in the reaction system is cut, trans-cutting activity of the Cas12a is activated, ssDNA in the reaction system is cut, and ssDNA-FQ is cracked. A full wavelength microplate reader was used to measure the fluorescence value of the detection reaction, with excitation wavelength of 485nm and emission wavelength of 520nm. The signal was recorded every 0.5 minutes for 0.5 hours.
In MERS virus gene fragment (MERS-upE) detection, lbCAs12a protein has higher fluorescence intensity and higher bypass cutting activity. In the novel Cas12a protein, cas12a-68 had weak bypass activity, and the fluorescent signal results at 5 minutes are shown in fig. 3. As shown in fig. 3, in the conventional detection system, the LbCpf1 protein can cleave the ssDNA fragment and release a fluorescent group, so that the detection system can be used for detecting MERS virus gene fragments, while Cas12a-68 protein can not cleave the ssDNA fragment in the system, and the detection reaction has almost no fluorescent signal, that is, cas 2a-68 can not detect MERS gene fragments in the conventional detection system.
3.Mn 2+ Effect of ions on Cras 12a protease cleavage Activity
In addition to conventional detection systems, we found Mn 2+ Can greatly improve the bypass cutting activity of protease. The detection system comprises 20 mu L of the following specific components: 200ng of purified Cas12a (Cas 12a-68 or LbCAs12 a), 25pM ssDNA-FQ, 1. Mu.M crRNA,10ng of dsDNA sample (MERS virus gene fragment) and a final volume of 20. Mu.L. The reaction was incubated at 37 ℃. The detection system is also added with a reaction buffer solution, wherein the reaction buffer solution comprises 100mM NaCl,50mM Tris-HCl,100 mug/mL BSA (bovine serum albumin) and the pH value is 7.5. 2. Mu.L of 10mM CaCl was added to the reaction buffer 2 、CoCl 2 、CuCl 2 、FeCl 2 、NiCl 2 、MgCl 2 、MnCl 2 、ZnCl 2 And EDTA, comparing the effect of different metal ions on Cas12a protease bypass activity.
The results are shown in FIG. 4, and the experimental results indicate that the Mn ratio is 2+ Under the condition, the protein activity is greatly improved, the activity of the Cas12a-68 protease is improved more than that of LBCas12a, and the fluorescence value of Cas12a-68 is improved by 55% relative to LbCAs12a in 5 minutes.
The fluorescence signals of both Cas12a and LbCas12a were elevated relative to the original reaction buffer 10×nebuffer r3.1, but the signal elevation of Cas12a-68 was greater. The results indicate that, in Mn 2+ In the presence, cas12-68 is able to detect the target molecule faster than LbCas12a, as well as a stronger fluorescent signal. In the subsequent experiments, mn is added into the Cas12a-68 protease reaction system 2+ 。
Example 2: effect of PAM on Cas12a-68 protease detection
According to step 3 in example 1, four nucleotide PAM sites, NNNN, each N having A, T, C, G possibilities, 64 total nucleic acids were synthesized, and forward and reverse sequence information was as follows:
MerS_F：ccattagtctctcNNNNgacatatggaaaacgaactatgttaccctttgtccaag，
MerS_R：cttggacaaagggtaacatagttcgttttccatatgtcNNNNgagagactaatgg，
the forward and reverse single-stranded sequences are synthesized respectively, the forward and reverse single-stranded sequences are dissolved to 100 mu M during synthesis, then 10 mu L of forward primer and 10 mu L of reverse primer corresponding to PAM are taken, 20 mu L of short-chain DNA is kept at the constant temperature of 95 ℃ for 2 minutes, then natural cooling is carried out to room temperature, and the single-stranded DNA is annealed and polymerized into double-stranded DNA.
The reaction buffer was 100mM NaCl,50mM Tris-HCl, 100. Mu.g/mL BSA (bovine serum albumin), pH 7.5, 10mM MnCl 2 . 200ng of purified Cas12a-68, 25pM ssDNA-FQ, 1. Mu.M crRNA,10ng dsDNA (i.e., MERS gene fragments containing different PAM) were added to each reaction, and the final reaction volume was 20. Mu.L. The reaction was carried out at 37℃for 15 minutes, and then the fluorescence signal value was read by a fluorescence detection instrument. Carrying out 64 fluorescence detection reactions, respectively reading fluorescence values of the reactions under different PAMs, and normalizing the values (namelyThe series of values was changed to fractions between 0 and 1, and fluorescence intensity heat maps were made for different PAMs.
The experimental results are shown in fig. 5, and Cas12a-68 can recognize not only PAM sites of TTTV of conventional Cas, but also various sites such as AGTN, GTTN, GCCN, GGCA and ATCT.
Example 3: detection of nucleic acid fragments in human gene fragments and human cell samples
1. Nucleic acid preparation
Preparation of nucleic acid: the crRNAs and Kras partial gene fragments and Kras gene fragments were synthesized by beijing, the biotechnology company limited, and the sequences are shown in tables 1 and 2.
TABLE 2
Preparation of human cells: human peripheral blood mononuclear cells were purchased from Shanghai bamboo hat Biotechnology Co., ltd, and mRNA was extracted. Then reverse transcription into cDNA, and extension of gene fragment with about 200bp length by Kras gene upper primer. The sequences of the primers on the Kras gene are shown in Table 3.
TABLE 3 Table 3
| Primer(s) | Sequence (5 '-3') |
| Mers_R | cacacataatctagtagccgtaagg |
| Mers_F | CCATGGGCtaccatgctg |
| Kras-F | ATGACTGAATATAAACTTGTG |
| Kras-R | GTCCCTCATTGCACTGTACTC |
2. Human gene fragment detection
At the G12C site of the Kras genome, the conventional Cas12a protease is undetectable because there is no recognition region for conventional PAM, such as no TTTV sequence. And our novel Cas12a-68 protease, in Mn 2+ The kit has higher reactivity in the presence and wider PAM recognition area, and can be used for detecting the Kras G12C locus.
As shown in FIG. 6, we designed 7 crRNA sequences, the sequence information of which is shown in Table 1. As can be seen from fig. 6, crRNA2 and crRNA5 can clearly distinguish wild-type and G12C mutated Kras gene fragments within 5min, whereas the corresponding PAMs of crRNA2 and crRNA5 are TTGC and GTTG, respectively, which are unrecognizable by conventional Cas12 proteases.
As shown in FIG. 6, lbCAs12a has no suitable PAM sequence, and the fluorescent signal is very weak and undetectable, both in the wild type and the G12C mutant. Using Cas12a-68 protease, crRNA1, crRNA3, crRNA4, crRNA6, crRNA7 could not distinguish between Kras wild type and G12C mutations, whereas crRNA2 and crRN5 could clearly distinguish between wild type and mutant. Cas12a-68 is demonstrated to detect sites that are undetectable by conventional Cas12 a.
3. Detection of human cell-derived gene fragments
In addition to in vitro PCR product detection of Kras fragment, mRNA is extracted from human peripheral blood mononuclear cells, reverse transcribed into cDNA, and then Kras fragment is specifically expanded, which is wild type and has no G12C mutation. The Kras fragment product in the cDNA was used for detection along with the in vitro Kras fragment.
As shown in FIG. 7, the test results show that the wild-type Kras fragment derived from the human cell and the Kras G12C mutation can be well distinguished at 5min by using Cas12a-68, crRNA2 and crRNA5 for experiments.
Example 4: specificity of Cas12a-68 protease detection
To determine the specificity of the detection of this system, we reacted the microRNAs of let7a and let7f, which were similar in sequence, according to the reaction conditions performed by the procedure of example 1. The double-stranded nucleotide sequence is shown in Table 2, and the difference between the two is only 1 base.
As a result of the reaction, as shown in FIG. 8, the reaction was conducted in Mn 2+ In the presence, let7a, which normally paired with crRNA (see Table 1), has a high fluorescence intensity, while the fluorescent signal at the position of let7f, which has a single base difference, is weak. However, even if crRNA and let7a are paired correctly, the conventional LbCpf1 protein has a low fluorescence signal, and cannot distinguish DNA fragments with a single base difference obviously. The Cas12-68 protease can accurately distinguish DNA fragments with 1 base difference.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (10)
- The application of Cpfl protein in preparing a kit for nucleic acid detection, wherein the amino acid sequence of the Cpf1 protein is shown as SEQ ID NO. 1.
- 2. A nucleic acid detection kit comprising a Cpf1 detection system, wherein said Cpf1 detection system comprises: guide RNA, cpf1 protein, nucleic acid probe and manganese ion-containing solution;the guide RNA is crRNA for guiding Cpf1 protein to specifically bind to nucleic acid;the amino acid sequence of the Cpf1 protein is shown as SEQ ID NO. 1.
- 3. The nucleic acid detection kit of claim 2, wherein the solution containing manganese ions is a manganese chloride solution;and/or, the nucleic acid probe is a single-stranded DNA probe;the single-stranded DNA probe comprises a fluorescent label;preferably, the single-stranded DNA probe has a fluorescent group and a fluorescence quenching group at its 5 'end and 3' end, respectively;preferably, the single-stranded DNA probe has a fluorescent group at its 5 'end and a fluorescence quenching group at its 3' end.
- 4. The nucleic acid detection kit of claim 3, wherein the fluorescent moiety is 6-FAM, TET, CY3, CY5, or ROX and the fluorescence quenching moiety is BHQ1, BHQ2, or BHQ3.
- 5. The nucleic acid detection kit of claim 2, wherein the Cpf1 detection system comprises the following specific components: 200ng Cpf1 protein, 25pM nucleic acid probe, 1. Mu.M crRNA, 2. Mu.L of sample to be tested, and a final volume of 20. Mu.L.
- 6. The nucleic acid detection kit of claim 2, further comprising a reaction buffer comprising NaCl, tris-HCl, and BSA; the pH of the reaction buffer was 7.5.
- 7. A method for detecting a nucleic acid of non-diagnostic interest, wherein the nucleic acid is detected using the Cpf1 detection system of any one of claims 2 to 6.
- 8. The assay of claim 7, wherein the reaction temperature of the Cpf1 assay is 37 ℃ and the reaction time of the Cpf1 assay is 5 minutes.
- 9. The method according to claim 8, wherein the nucleic acid in the sample to be tested is released by a nucleic acid quick-release reagent;the detection method further comprises the step of reading the result, and the result is read by naked eyes under an enzyme-labeled instrument or a fluorescent lamp.
- 10. The method of detecting according to claim 7, wherein the specific steps include:(1) Acquiring DNA of a sample to be detected;(2) Detecting by using a detection system, wherein the detection system reacts for 5 minutes at 37 ℃; the detection system comprises the following specific components: 200ng Cpf1 protein, 25pM nucleic acid probe, 1. Mu.M crRNA,10ng DNA of the sample to be tested, and a final volume of 20. Mu.L; reaction buffer 100mM NaCl,50mM Tris-HCl, 100. Mu.g/mL BSA,10mM MnCl 2 ，pH 7.5；(3) And (3) detecting the fluorescence value in the detection system in the step (2) by using an enzyme-labeled instrument.
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