A kind of Mb2Cas12a-RRVRR mutant protein and its preparation method and application

Abstract

The present invention belongs to the field of gene editing and relates to Cas12a proteins, in particular to an Mb2Cas12a-RRVRR mutant protein and its preparation method and application. This study evaluated 16 Cas12a proteins for the first time, especially their cleavage activity at lower temperatures. Moraxella bovoculi The activity of the Mb2Cas12a protein of strain 57922 is stronger than that of other proteins. In addition, the present application introduces 5 mutations into the Mb2Cas12a protein to create the Mb2Cas12a-RRVRR variant protein, which has a wider range of PAMs and stronger activity. In summary, the new detection tool based on CRISPR / Mb2Cas12a-RRVRR variant enhances the diagnostic capability of CRISPR technology and has the potential to replace the existing Cas12a protein.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing and relates to a Cas12a protein, and in particular refers to an Mb2Cas12a-RRVRR mutant protein and a preparation method and application thereof. Background Art

[0002] In recent years, diagnostic systems based on Crispr / Cas nucleases have been widely used in the detection of crop pathogens. Among them, most of the developed detection technologies mainly rely on the principle that Cas proteins such as Cas12 and Cas13a are assembled into binary complexes with crRNA. After specifically recognizing the detection target, they will arbitrarily cut the adjacent single-stranded DNA / RNA nucleic acid reporter molecules. An integrated CRISPR / Cas detection platform requires 4 steps, namely target pre-amplification, target recognition by Cas / crRNA complex, Cas protein activation and reporter molecule cleavage. Cas12a can be activated by dsDNA / ssDNA targets and non-specifically cut ssDNA, while Cas13 can be activated by ssRNA targets and cleave ssRNA reporter molecules. By programming the sequences recognized by Cas / crRNA, various diagnostic systems such as SHERLOCK and DETECTR have been developed and widely used in the detection of viruses, pathogenic microorganisms and other small molecules.

[0003] Although the existing CRISPR / Cas12 detection system is very sensitive and can achieve aM detection level in combination with isothermal amplification technology, there is still potential for further improvement in methodology. Early CRISPR diagnostic platforms usually take 30 minutes or more, but on-site detection requires shortening the waiting time. In addition, the Cas12a protein needs to be heated at 37°C to effectively maintain protein activity, which may cause inconvenience to on-site applications. Another limitation is that the current Cas12a protein prefers to recognize the conventional TTTV (V=A, C, or G) PAM (Protospacer adjacent motif) sequence and shows very low activity in recognizing other unconventional PAMs, which limits the scope of crRNA. So far, only a few Cas12a proteins have been evaluated and used in the development of detection systems. However, Cas proteins from different microbial strains have different recognition efficiencies and cleavage activities. Therefore, screening new Cas12a proteins is an important means to establish more effective CRISPR / Cas diagnostic tools; Patent 201811588275.6 discloses a CRISPR / Cas12a gene editing system and its application, which screens LiCas12a proteins with endonuclease activity from microorganisms and establishes a CRISPR / LiCas12a gene editing system for gene editing in mammals and Escherichia coli; Patent 201911106924.9 discloses a Cas12a protein that recognizes TTTV and TTV double PAM sites. Although the protein can recognize TTV PAM sites, the temperature of the recognition system is high; in order to further find new Cas12a proteins that can target relaxed PAMs and further speed up the detection process, especially at lower ambient temperatures. This research group has conducted long-term exploratory research. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the present invention proposes a Mb2Cas12a-RRVRR mutant protein and a preparation method and application thereof.

[0005] The technical solution of the present invention is achieved in this way:

[0006] A Mb2Cas12a-RRVRR mutant protein, wherein the Mb2Cas12a-RRVRR mutant protein is obtained by mutation of amino acids at positions 172, 563, 569, 573, and 625 of the Mb2Cas12a protein.

[0007] Furthermore, the amino acids at positions 172, 569, 573 and 625 are all mutated to R, and the amino acid at position 563 is mutated to V.

[0008] Further, the Mb2Cas12a protein is selected from Moraxella bovoculi strains.

[0009] Furthermore, the amino acid sequence of the Mb2Cas12a-RRVRR mutant protein is shown in SEQ ID No.2.

[0010] Gene sequence encoding the above-mentioned Mb2Cas12a-RRVRR mutant protein.

[0011] The nucleotide sequence of the above gene sequence is shown in SEQ ID No.1.

[0012] The preparation method of the above-mentioned Mb2Cas12a-RRVRR mutant protein comprises the following steps:

[0013] (1) The Cas12a coding sequence of the Mb2Cas12a gene was codon optimized, and then the nucleotides at positions 514 and 515 were mutated to CG, the nucleotides at positions 1687 and 1688 were mutated to CG, the nucleotides at positions 1705 and 1706 were mutated to GT, the nucleotides at positions 1717 and 1718 were mutated to CG, and the nucleotides at positions 1874 were mutated to G to obtain the mutated Mb2Cas12a-RRVRR gene sequence;

[0014] (2) Inserting the mutated Mb2Cas12a-RRVRR gene sequence into the pET-N-His-TEV vector to obtain the pET-Mb2Cas12a-RRVRR vector;

[0015] (3) The pET-Mb2Cas12a-RRVRR vector was transferred into Escherichia coli BL21, and after culture and induction of expression, the target protein, i.e., the Mb2Cas12a-RRVRR mutant protein, was centrifuged, lysed, and purified.

[0016] Furthermore, the Mb2Cas12a gene sequence in step (1) is shown in SEQ ID No.3.

[0017] Furthermore, the mutated Mb2Cas12a-RRVRR gene sequence in step (1) is as shown in SEQ ID No. 1.

[0018] Application of the above-mentioned Mb2Cas12a-RRVRR mutant protein in the detection of genetically modified crops.

[0019] The present invention has the following beneficial effects:

[0020] 1. This study evaluated 16 Cas12a proteins for the first time, especially their cleavage activity at lower temperatures. We found that Moraxella bovoculiThe Mb2Cas12a protein of strain 57922 is more active than other proteins. In addition, we introduced five mutations into the Mb2Cas12a protein to create a variant protein (Mb2Cas12a-RRVRR) with a wider range of PAMs and stronger activity. In summary, the new detection tool based on CRISPR / Mb2Cas12a-RRVRR variant enhances the diagnostic capabilities of CRISPR technology and has the potential to replace the existing Cas12a protein.

[0021] 2. The reaction temperature of the Mb2Cas12a-RRVRR mutant protein of the present application is 30°C, and the sensitivity of the Mb2Cas12a-RRVRR protein in detecting the target is 2.4 × 10 3 The sensitivity of Mb2Cas12a-RRVRR protein to detect targets is 24 copies / reaction, which is 10 times higher than that of the commercialized LbCas12a protein. At a reaction temperature of 35°C, the sensitivity of Mb2Cas12a-RRVRR protein to detect targets is 24 copies / reaction, which is much higher than that of LbCas12a protein. Therefore, Mb2Cas12a-RRVRR can be developed into a more sensitive Crispr / Cas12a diagnostic tool. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 Comparison of the promiscuous cleavage activities of 16 Cas12a protein families.

[0024] Figure 2 Mb2cas12a has stronger random cutting activity at low temperatures; (A) The activity differences of 16 Cas12a proteins at different temperatures; (B) Mb2Cas12a is more active than the commercialized LbCas12a protein at low temperatures.

[0025] Figure 3Comparison of the in vitro random cleavage activity of Mb2Cas12a and its RRVRR variant proteins on dsDNA substrates with different PAM sequences; (A) In vitro PAM identification test scheme design; PAM library contains 256 47BP-long dsDNA fragments, composed of 4 random nucleotides, synthesized by annealing complementary ssDNA. Only one dsDNA substrate was used as the target in each analysis; Heat map describes the fluorescence intensity of wild-type Mb2Cas12a (B) and its RRVRR variant protein (C).

[0026] Figure 4 Comparison of the sensitivity of four Cas12a proteins in RPA / Cas12a detection; (A) reaction temperature is 30°C, (B) reaction temperature is 35°C.

[0027] Figure 5 The difference in the enzymatic activity of the two Cas12a proteins in catalyzing trans-cleavage. The dsDNA activators have the same targeting sequence. The solid line fits the Michaelis-Menten equation. The calculated values ​​of the activity parameters are given in the table below the figure. The data are shown as mean ± standard deviation (n=3). DETAILED DESCRIPTION

[0028] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] 1. Mb2Cas12a has stronger promiscuous cutting activity

[0031] Sixteen Cas12a proteins were selected from the Cas12a family, including three commercialized Cas12a nucleases (FnCas12a, AsCas12a, and LbCas12a), and 13 Cas12a homologs that have not yet been evaluated for trans-cleavage activity. Figure 1As shown in A, these Cas12a proteins from different bacteria have low homology at the amino acid level. For all Cas12a proteins, the PAM sequences of TTTA and TTTG in dsDNA targets can induce protein trans-cleavage activity, while dsDNA targets without typical PAMs can hardly activate the activity of Cas12a proteins. Nine Cas12a homologs (As, Er, Lb, Mb2, MI, Fn, Ts, Px and Lp) completely degraded fluorescently labeled ssDNA reporters within 30 minutes and showed sufficient cleavage activity in vitro, among which Mb2Cas12a from Moraxella bovoculi strain 57922 had the highest trans-cleavage activity among Cas12a homologs and was able to recognize the PAM sequence of GCTC ( Figure 1 B).

[0032] At different temperatures of 24-48°C, 16 Cas12a proteins showed different levels of cleavage efficiency, and it was found that the optimal reaction temperature of these proteins was around 40 ~ 44°C ( Figure 2 A). Reaction temperatures below 30°C significantly inhibit the protein's random cutting activity, but Mb2Cas12a is more tolerant to low temperatures and can function fairly stably even at 24°C and 28°C. At relatively low reaction temperatures, Mb2Cas12a showed higher enzyme activity than the commercial LbCas12a ( Figure 2 B). Therefore, Mb2Cas12a may be a better alternative nuclease for Crispr / Cas12a diagnostic system.

[0033] 2. Mb2Cas12a-RRVRR mutant protein has a wider PAM sequence preference

[0034] To evaluate whether the Mb2Cas12a-RRVRR variant could recognize dsDNA targets with a wider range of PAMs, its promiscuous cleavage activity was measured using an in vitro PAM recognition assay.

[0035] Design of in vitro PAM identification assay. The PAM library contains 256 47bp dsDNA fragments, which are synthesized by annealing base complementary pairing of the positive strand (5'-ACTCAATTGTNNNNGCATACATGGCATTGCACATATCACTACAGTCA-3') and the complementary strand (3'-TGAGTTAACANNNNCGTATGTACCGTAACGTGTATAGTGATGTCAGT-5'), where NNNN is the PAMs sequence, which consists of four random nucleotides A / T / G / C. Only one dsDNA substrate was used as the target for each analysis, and finally 256 pairs of 47bp long base complementary oligonucleotide sequences were synthesized. These dsDNA substrates share the same target sequence but different PAMs ( Figure 3 A).

[0036] The reaction system was composed of 100 nM Cas12a protein, 120 nM crRNA, 400 nM ssDNA FQ-reporter (5′-FAM-TTATT-Quencher-3′, synthesized by Sangon Biotechnology), 20 U RNase inhibitor, 100 nM dsDNA target fragment, and 5 μL NEBuffer 3.1 (10×), and ddH2O was added to 50 μL for trans-cleavage activity assay.

[0037] The reaction was carried out at 37 ºC on a Bio-Tek FLx800 microplate fluorescence reader, and fluorescence was measured every 30 s (λ ex: 485 nm; λ em: 535 nm). By monitoring the fluorescence intensity in the reaction system, we found that the wild-type Mb2Cas12a has a high tolerance for changes in the 4th base and can reliably bind to the NTTV PAMs target ( Figure 3 B). Compared with the wild-type Mb2Cas12a protein, the RRVRR variant of Mb2Cas12a can recognize the PAMs sites of NTTV, NTCV and NCTV and exhibits stronger promiscuous cutting activity ( Figure 3 C).

[0038] 3. Mb2Cas12a-RRVRR mutant protein has higher sensitivity at lower temperatures

[0039] Next, we evaluated the sensitivity of the novel Mb2Cas12a-RRVRR mutant protein in detecting target nucleic acids. The reaction was based on the above system with the following adjustments: the dsDNA target concentration was diluted to 1 aM~10 nM, and 2 μL was added, which was equivalent to 2.4~2.4×10 10copies / reaction, and the fluorescence value in the system is detected after 15 minutes of reaction. Figure 4 As shown in Figure 2, we found that at a lower temperature, such as a reaction temperature of 30°C, the sensitivity of Mb2Cas12a-RRVRR protein to detect the target was 2.4 × 10 3 The sensitivity of Mb2Cas12a-RRVRR protein to detect targets is 24 copies / reaction, which is 10 times higher than that of the commercialized LbCas12a protein. At a reaction temperature of 35°C, the sensitivity of Mb2Cas12a-RRVRR protein to detect targets is 24 copies / reaction, which is much higher than that of LbCas12a protein. Therefore, Mb2Cas12a-RRVRR can be developed into a more sensitive Crispr / Cas12a diagnostic tool.

[0040] Example 2

[0041] 1. Prokaryotic expression and mutant construction

[0042] The Cas12a coding sequence of Moraxella bovoculi 57922 strain (Mb2Cas12a, NCBI accession number WP_046697655.1) was codon-optimized and synthesized using GenScript (GenWeichi, Nanjing). Then, the nucleotides at positions 514 and 515 were mutated to CG, the nucleotides at positions 1687 and 1688 were mutated to CG, the nucleotides at positions 1705 and 1706 were mutated to GT, the nucleotides at positions 1717 and 1718 were mutated to CG, and the nucleotides at positions 1874 were mutated to G, and the mutated Mb2Cas12a-RRVRR gene sequence was shown in SEQ ID No. 1, and then the mutated Mb2Cas12a-RRVRR gene sequence and the unmutated Mb2Cas12a gene sequence were inserted into the pET-N-His-TEV vector to construct Mb2Cas12a-RRVRR (D172R, N563R, K569V, N573R and K625R) and pET-Mb2Cas12a prokaryotic expression vectors, respectively.

[0043] Both vectors were transformed into Escherichia coli BL21 (DE3) and cultured in liquid Terrific Broth (TB) medium at 37°C overnight. The above bacterial suspension was inoculated into 2 L of fresh TB medium and further cultured at 37°C and 200 rpm until the OD600 was approximately 0.8. Subsequently, 0.4 mM IPTG was added and protein expression was induced at 16°C for 12 h. After centrifugation (4°C, 6 000×g, 20 min), the resulting cell pellet was resuspended in 20 ml lysis buffer [50 mMTris-HCl, 150 mM NaCl, 1× final concentration of protease inhibitor (Clontech), 0.5 mg / ml lysozyme] and then sonicated in ice water. The supernatant after centrifugation was further purified using the His-tag Protein Purification Kit (Beyotime Biotechnology, China). The target protein was washed with 20 mM imidazole and eluted again with 300 mM imidazole buffer (50 mM Tris, 150 mM NaCl, pH 8.0). The soluble Cas12a protein was incubated with TEV protease (His-tag) at 4 °C and dialyzed overnight to remove the N-terminal His-tag. After concentration with a Pierc protein concentrator PES (ThermoScientific), the protein was further purified using a fast protein liquid chromatography (GE AKTA Purifier 100 / 10) equipped with a HiLoad 16 / 600 Superdex 200 pg column (GE HealthcareLife Sciences) and eluted with storage buffer (200 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 0.1 mg / ml BSA, pH 7.9). The protein concentration was determined, diluted to 5 μM, and stored at -80 °C for future use.

[0044] 2. Mb2Cas12a-RRVRR mutant protein has a wider PAM sequence preference

[0045] We introduced five mutations into the Mb2Cas12a protein to create a Mb2Cas12a variant protein (Mb2Cas12a-RRVRR). To evaluate whether the Mb2Cas12a-RRVRR variant can recognize dsDNA targets with a wider range of PAMs, its promiscuous cleavage activity was measured using an in vitro PAM recognition assay.

[0046] Design of in vitro PAM identification assay. The PAM library contains 256 47bp dsDNA fragments, which are synthesized by annealing base complementary pairing of the positive strand (5'-ACTCAATTGTNNNNGCATACATGGCATTGCACATATCACTACAGTCA-3') and the complementary strand (3'-TGAGTTAACANNNNCGTATGTACCGTAACGTGTATAGTGATGTCAGT-5'), where NNNN is the PAMs sequence, which consists of four random nucleotides A / T / G / C. Only one dsDNA substrate was used as the target for each analysis, and finally 256 pairs of 47bp long base complementary oligonucleotide sequences were synthesized. These dsDNA substrates share the same target sequence but different PAMs ( Figure 3 A).

[0047] The reaction system was composed of 100 nM Cas12a protein, 120 nM crRNA, 400 nM ssDNA FQ-reporter (5′-FAM-TTATT-Quencher-3′, synthesized by Sangon Biotechnology), 20 U RNase inhibitor, 100 nM dsDNA target fragment, and 5 μL NEBuffer 3.1 (10×), and ddH2O was added to 50 μL for trans-cleavage activity assay.

[0048] The reaction was carried out at 37 ºC on a Bio-Tek FLx800 microplate fluorescence reader, and fluorescence was measured every 30 s (λ ex: 485 nm; λ em: 535 nm). By monitoring the fluorescence intensity in the reaction system, we found that the wild-type Mb2Cas12a has a high tolerance for changes in the 4th base and can reliably bind to the NTTV PAMs target ( Figure 3 B). Compared with the wild-type Mb2Cas12a protein, the RRVRR variant of Mb2Cas12a can recognize the PAMs sites of NTTV, NTCV and NCTV and exhibits stronger promiscuous cutting activity ( Figure 3 C).

[0049] 3. Mb2Cas12a-RRVRR mutant protein has higher sensitivity at lower temperatures

[0050] Next, we evaluated the sensitivity of the novel Mb2Cas12a-RRVRR mutant protein in detecting target nucleic acids. The reaction was based on the above system with the following adjustments: the dsDNA target concentration was diluted to 1 aM~10 nM, and 2 μL was added, which was equivalent to 2.4~2.4×10 10copies / reaction, and the fluorescence value in the system is detected after 15 minutes of reaction. Figure 4 As shown in Figure 2, we found that at a lower temperature, such as a reaction temperature of 30°C, the sensitivity of Mb2Cas12a-RRVRR protein to detect the target was 2.4 × 10 3 The sensitivity of Mb2Cas12a-RRVRR protein to detect targets is 24 copies / reaction, which is 10 times higher than that of the commercialized LbCas12a protein. At a reaction temperature of 35°C, the sensitivity of Mb2Cas12a-RRVRR protein to detect targets is 24 copies / reaction, which is much higher than that of LbCas12a protein. Therefore, Mb2Cas12a-RRVRR can be developed into a more sensitive Crispr / Cas12a diagnostic tool.

[0051] 4. Activity evaluation of Mb2Cas12a and Mb2Cas12a-RRVRR mutant proteins

[0052] Michaelis-Menten analysis uses low concentrations of Mb2Cas12a, crRNA and dsDNA targets for reaction, and the final concentrations are fixed at 10 nM, 12 nM and 0.1 nM, respectively. The reaction is carried out using a fluorescence detection microplate reader, and the total system is 50 microliters, including 10 nM Cas12a protein, 12 nM crRNA, 0.1 nM dsDNA target sequence, different concentrations of FQ-reporter (0, 10 nm, 100 nm, 200 nm, 400 nm, 600 nm, 800 nm, 1000 nm and 1500 nm), 20 U RNase inhibitor, 5 μL NEBuffer 3.1 (10×) to form the reaction system and add ddH2O to 50 μL for trans-cleavage activity determination. Cas12a protein and crRNA were pre-mixed and pre-incubated at 25 °C for 10 min to promote the formation of RNA / Cas12a complex. The reaction was carried out at 37 ºC on a Bio-Tek FLx800 microplate fluorescence detector, and fluorescence was measured every 30 s (λ ex: 485 nm; λ em: 535 nm). GraphPad Prism 8 software was used to calculate the fluorescence intensity, including K m and K cat The enzyme kinetic parameters.

[0053] Through Michaelis-Menten kinetic analysis, we observed that the catalytic efficiency of Mb2Cas12a-RRVRR (expressed as Kcat / Km value) was 1.34 times that of Mb2Cas12a ( Figure 5). These results indicate that mutations of these amino acids in Mb2Cas12a-RRVRR can significantly improve its activity against the TTTV PAM target to a certain extent, retaining a more potent trans-cleavage activity.

[0054] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Mb2Cas12a-RRVRR mutant protein, characterized in that: The Mb2Cas12a-RRVRR mutant protein is obtained by mutation of amino acids at positions 172, 563, 569, 573, and 625 of the Mb2Cas12a protein; The amino acids at positions 172, 569, 573 and 625 are all mutated to R, and the amino acid at position 563 is mutated to V; The Mb2Cas12a protein is selected from Moraxella bovoculi strain; The amino acid sequence of the Mb2Cas12a-RRVRR mutant protein is shown in SEQ ID No.

2.

2. The gene sequence encoding the Mb2Cas12a-RRVRR mutant protein according to claim 1, wherein the nucleotide sequence is as shown in SEQ ID No.

1.

3. The method for preparing the Mb2Cas12a-RRVRR mutant protein according to claim 1, characterized in that Here are the steps: (1) The gene coding sequence of the Mb2Cas12a protein was codon optimized, and then the nucleotides at positions 514 and 515 were mutated to CG, the nucleotides at positions 1687 and 1688 were mutated to CG, the nucleotides at positions 1705 and 1706 were mutated to GT, the nucleotides at positions 1717 and 1718 were mutated to CG, and the nucleotides at positions 1874 were mutated to G to obtain the mutated Mb2Cas12a-RRVRR gene sequence; (2) Inserting the mutated Mb2Cas12a-RRVRR gene sequence into the pET-N-His-TEV vector to obtain the pET-Mb2Cas12a-RRVRR vector; (3) The pET-Mb2Cas12a-RRVRR vector was transferred into Escherichia coli BL21, and after culture and induction of expression, the target protein, i.e., the Mb2Cas12a-RRVRR mutant protein, was centrifuged, lysed, and purified; The Mb2Cas12a gene sequence in step (1) is shown in SEQ ID No.

3.

4. The method for preparing the Mb2Cas12a-RRVRR mutant protein according to claim 3, characterized in that: The mutated Mb2Cas12a-RRVRR gene sequence in step (1) is shown in SEQ ID No.

1.

5. Application of the Mb2Cas12a-RRVRR mutant protein described in claim 1 in genetically modified crop detection.

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