A modification of LbCas12a enzyme to improve thermal stability and its application

Abstract

The present invention relates to a LbCas12a artificially modified enzyme with improved thermal stability, the amino acid sequence of the LbCas12a artificially modified enzyme is shown in SEQ ID NO.1, and the nucleotide gene sequence encoding the LbCas12a artificially modified enzyme is shown in SEQ ID NO.2. Compared with the original LbCas12a enzyme, the mutant Y554C obtained by screening the present invention has a thermal stability improved by about 8 ° C, which not only meets the reaction requirements in the CRISPR / Cas12a system, but also improves the application range and use effect of the LbCas12a enzyme, and can be used for commercial applications.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and in particular relates to an LbCas12a artificially modified enzyme with improved thermal stability and an application thereof. Background Art

[0002] The Cas12a protein belongs to the second type of CRISPR / Cas V system. Compared to the Cas9 system, which has long been used for gene editing, the Cas12a system is a single RNA-guided endonuclease. The Cas12a system uses a unique mechanism to recognize target DNA. The Cas12a protein has specific RNA cleavage activity, meaning that it can process the primary transcript pre-crRNA into mature crRNA without the need for additional transactivating crRNA (tracrRNA). Therefore, multiple crRNA sequences can be connected in series to achieve multi-target gene editing on a single vector.

[0003] The CRISPR / Cas12a system has great application potential in the field of molecular detection, among which the LbCas12a protein is one of the hot spots of application. By utilizing the trans-cleavage characteristics of the LbCas12a protein and combining it with a fluorescently labeled single-stranded nucleic acid probe, highly sensitive detection can be achieved. When detecting samples, the CRISPR / Cas12a system is usually combined with isothermal amplification. In order to avoid low specificity and contamination, it is necessary to combine high-temperature isothermal amplification to avoid the problem of poor detection specificity. In addition, combining isothermal amplification with the CRISPR / Cas12a system into a one-pot reaction system can avoid contamination. Since the optimal reaction temperature of the LbCas12a enzyme is 37°C, in a one-pot system, the temperature tolerance of the LbCas12a enzyme needs to be improved to avoid its inactivation and affect the reaction.

[0004] Therefore, modifying the LbCas12a protease from the biomolecular level to obtain an artificially modified enzyme with good stability is one of the current research and development directions in this field. Summary of the Invention

[0005] To solve the above technical problems, the present invention includes the following aspects:

[0006] The first aspect of the present invention provides a LbCas12a artificially modified enzyme with improved thermal stability, the amino acid sequence of which is shown in SEQ ID NO.1:

[0007]

[0008] The second aspect of the present invention provides a LbCas12a artificial modification enzyme nucleotide gene, which encodes the above-mentioned thermostable LbCas12a artificial modification enzyme, and the nucleotide gene sequence is shown in SEQ ID NO.2:

[0009]

[0010] A third aspect of the present invention provides a recombinant expression vector comprising the above-mentioned LbCas12a artificially modified enzyme nucleotide gene.

[0011] Preferably, the recombinant expression vector is pET-28a(+).

[0012] The fourth aspect of the present invention provides a host cell, which comprises the above-mentioned recombinant expression vector.

[0013] Preferably, the host cell is Escherichia coli, and more preferably, the Escherichia coli is Escherichia coli BL21.

[0014] A fifth aspect of the present invention provides the use of the above-mentioned thermostable LbCas12a artificially modified enzyme in the CRISPR / Cas12a system reaction.

[0015] The technical effects produced by the present invention are:

[0016] 1. The present invention connects the new artificially modified LbCas12a enzyme gene with a shuttle expression plasmid to form a recombinant expression plasmid, and then transforms the recombinant expression plasmid into a prokaryotic cell expression protein and integrates it into the prokaryotic cell genome to obtain recombinant Escherichia coli. The recombinant Escherichia coli is cultured and the expression of the modified LbCas12a enzyme is induced to obtain a stable LbCas12a artificially modified enzyme.

[0017] 2. Based on the study of the thermodynamic structure of pepsin, the present invention screens LbCas12a enzyme mutants and obtains mutant Y554C (named CAIQ-Cas12a enzyme) with good activity at 54 ° C. Compared with the original LbCas12a enzyme, the mutant of the present invention has a thermal stability increased by about 8 ° C, which not only meets the reaction requirements in the CRISPR / Cas12a system, but also improves the application range and use effect of the LbCas12a enzyme, and can be used for commercial applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is an agarose gel electrophoresis diagram of colony PCR verification of product size accuracy in Example 1 of the present invention, wherein lane M is a marker and lanes 1-20 are 20 single colony samples picked;

[0019] Figure 2 This is an agarose gel electrophoresis diagram of double enzyme digestion verification of the plasmid extracted after expansion culture of a single colony with correct PCR bands from three colonies in Example 1 of the present invention, wherein lane M is a marker; lanes 1, 3, and 5 are double enzyme digestion results, and lanes 2, 4, and 6 are intact plasmid results;

[0020] Figure 3 The protein expression results of the mutants in Example 3 of the present invention are shown, where M is a marker, lane 1 is Y554C, lane 2 is the original LbCas12a enzyme, and lane 3 is a blank control;

[0021] Figure 4 This is the result of verifying the cleavage activity of the mutant Y554C (CAIQ-Cas12a enzyme) at different temperatures in Example 4 of the present invention, where AG corresponds to reaction temperatures of 37°C, 46°C, 48°C, 50°C, 52°C, 54°C, and 56°C, respectively; lanes 1-3 are mutant Y554C, original enzyme, and blank control, respectively;

[0022] Figure 5 Comparison of fluorescence values ​​of mutant Y554C (CAIQ-Cas12a enzyme) and the original enzyme after reaction at different temperatures in Example 5 of the present invention;

[0023] Figure 6 The results of the cleavage activity verification of the mutant K20F in Comparative Example 1 of the present invention at different temperatures are shown, where AG corresponds to reaction temperatures of 37°C, 46°C, 48°C, 50°C, 52°C, 54°C, and 56°C, respectively. Lanes 1-3 represent the mutant K20F, the original enzyme, and the blank control, respectively.

[0024] Figure 7 It is the enzyme activity detection result of the original LbCas12a enzyme at different temperatures in Comparative Example 2 of the present invention, wherein lanes 1-12 are 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, and 48°C, respectively; DETAILED DESCRIPTION

[0025] The present invention predicts LbCas12a enzyme mutants through the results of Hotspot Wizard, Average B-Factors and I-mutant stability software, screens mutants according to the unfolding free energy ΔΔG and B factor in combination with thermophilic amino acid preference, and performs mutation based on the above results to effectively improve the thermostability of the enzyme.

[0026] Example 1. Construction of mutant plasmid

[0027] Plasmid pET-28(a)-LbCas12a was maintained in the laboratory. PCR amplification was performed using a two-step RF cloning method to introduce mutations. The specific primers are as follows:

[0028] Upstream primer: 5′-GCAGCAAATACTGCCTGGCTATCA-3′ (SEQ ID NO. 3);

[0029] Downstream primer: 5′-CGGCTGTTTTTGAAACCCGAG-3′ (SEQ ID NO. 4).

[0030] RF1 reaction system: plasmid pET-28 (a) -LbCas12a template 50 ~ 150ng; upstream primer, 10μM, 0.5 ~ 2μL; downstream primer, 10μM, 0.5 ~ 2μL; PrimeSTAR DNA polymerase, 2.5U / μL, 0.5 ~ 1.5μL; 5 × PrimeSTAR Buffer (containing 10mmol / Lmg 2+ ), 5μL; dNTPs, 2.5mM, 0.5~2μL; add sterile double-distilled water to a total volume of 25μL.

[0031] The amplification reaction was carried out on a PCR instrument. The reaction procedure for RF1 was as follows:

[0032] Pre-denaturation at 94°C for 5 min; 30 cycles of denaturation at 94°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 1 min; then continue extension at 72°C for 5 min and cool to 4°C.

[0033] The product of RF1 is purified and recovered as the primer for RF2. The specific reaction system of RF2 is as follows:

[0034] Plasmid pET-28(a)-LbCas12a template 50-150 ng; RF1 product 100-200 ng; PrimeSTAR GXL DNA polymerase, 2.5 U / μL, 0.5-1.5 μL; 5× PrimeSTAR GXL Buffer (containing 10 mmol / L Mg 2+ ), 5 μL; dNTPs, 2.5 mM, 0.5-2 μL; sterile double-distilled water was added to a total volume of 25 μL.

[0035] The amplification reaction was carried out on a PCR instrument. The reaction procedure for RF2 was as follows:

[0036] Denaturation at 98°C for 10 s, annealing at 60°C for 15 s, and extension at 68°C for 8 min 5 s were performed for 30 cycles; the temperature was then lowered to 4°C.

[0037] The obtained RF2 product was digested with DpnⅠ enzyme at 37℃ for 3h, and the digestion product was transformed into competent cells E.coliDH5α. The transformed cells were spread on a resistant solid LB medium plate and inverted for 12h. Twenty single colonies were picked and colony PCR was performed using universal primers of pET-28a(+) plasmid, as shown in Figure 2. Figure 1As shown. Three bacteria with correct band size and no non-specific amplification were selected for expansion culture and plasmid extraction. Double enzyme digestion with ScaⅠ and XbaⅠ was used to verify the correctness of the plasmid size, as shown. Figure 2 As shown, the plasmid with the correct band after enzyme digestion was sequenced, and the correct sequence could be used for subsequent experiments.

[0038] Example 2: Construction of expression strain

[0039] The mutant plasmid constructed in Example 1 was transformed into competent E. coli BL21 cells, and the competent cells were immediately placed in an ice bath and waited for the cells to thaw; under a sterile operating environment, 2 μL of mutant plasmid DNA was gently mixed with 100 μL of freshly thawed competent cells and allowed to stand on ice for 30 min; the competent cells were heat shocked at 42° C. for 45 s and immediately placed in an ice bath for 5 min; 5 mL of LB liquid medium (0.5% (m:V) yeast powder, 1.0% (m:V) peptone, 1.0% (m:V) NaCl, dissolved in ultrapure water, sterilized at 121° C. for 20 min) was added and incubated at 37° C. for 1 h; centrifuged at 6000 × g for 1 min, and 70-100 μL of liquid was retained to resuspend the cells. The bacterial solution was evenly spread on an LB solid plate (containing 1.5% (m:V) agar and 50 μg / mL Kana) and incubated inverted in a 37° C. constant temperature incubator overnight. Pick a single colony and add it to 5 mL of LB liquid medium as a seed solution, and incubate at 37°C for 12 h. To expand the strain, transfer the seed solution to 200 mL of LLB liquid medium (0.5% (m:V) yeast extract, 1.0% (m:V) peptone, 0.5% (m:V) NaCl, dissolved in ultrapure water, sterilized at 121°C for 20 min) and incubate at 37°C for 2 h.

[0040] Example 3. Expression of mutant Y554C

[0041] The inducer IPTG was added to the bacteria after the expanded culture in Example 2 at a final concentration of 0.5-0.8 mM. After induction at 16°C for 16-18 hours, the bacteria were harvested and centrifuged at 8000g and 4°C, the supernatant was removed, and sterile double-distilled water was added for resuspending; the supernatant was removed at 8000g and 4°C, 1×PBS was added for resuspending; the supernatant was removed at 8000g and 4°C, 30 mL of 1×PBS was added for resuspending. Ultrasonic lysis was performed on ice at an ultrasonic power of 300W for 3 seconds, and the solution was allowed to stand for 5 seconds. This cycle was repeated several times until the solution was basically clear. Centrifuged at 4°C and 10000g for 20 minutes, 10 μL of supernatant protein sample was taken for 12% SDS-PAGE polyacrylamide gel electrophoresis detection. The results are as follows. Figure 3 As shown, the target band was found at 135 kDa, indicating that the mutant was successfully expressed.

[0042] Example 4: Enzyme activity detection of mutant Y554C at different temperatures

[0043] The mutant Y554C was reacted at 37℃, 46℃, 48℃, 50℃, 52℃, 54℃ and 56℃ respectively to detect its enzyme activity at different temperatures. Figure 5 As shown, the mutant Y554C still has cutting activity at 50°C, and its thermal stability is about 8°C higher than that of the original enzyme, and can be used for commercial applications in the future.

[0044] Example 5. Comparison of fluorescence values ​​of the Y554C mutant and the original enzyme after reaction at different temperatures

[0045] The fluorescence values ​​of the original enzyme and the Y554C mutant at the optimal reaction temperatures of 37°C and 50°C for the original enzyme were compared (see Figure 6 The results showed that at 37°C, the fluorescence value after the reaction with the original enzyme was 4107.67 au, while the fluorescence value after the reaction with the Y554C mutant was 4172.33 au, indicating that the mutant still had strong cleavage activity at the optimal reaction temperature of the original enzyme. At 50°C, the fluorescence value after the reaction with the original enzyme was only 986 au, which was not visible to the naked eye. However, the fluorescence value after the reaction with the Y554C mutant was 3585.33 au, which was visible to the naked eye, indicating that the Y554C mutant had strong cleavage activity.

[0046] Comparative Example 1: Thermal stability test of mutant K20F

[0047] Comparative Example 1 provides a mutant of an LbCas12a enzyme. The results of Hotspot Wizard, Average B-Factors, and I-mutant stability software are used to predict the LbCas12a enzyme mutant. The mutant is constructed according to the unfolding free energy ΔΔG and the B factor in combination with the thermophilic amino acid preference. The only difference from Example 1 is that the upstream primer and the downstream primer used are different. The primers used in Comparative Example 1 include:

[0048] Upstream primer: 5′-CTGCGCTTCTTCGCGATCCCG-3′ (SEQ ID NO. 5);

[0049] Downstream primer: 5′-AGTTACGGATTGCGTCGTAGATGTG-3′ (SEQ ID NO. 6).

[0050] The construction of the expression strain and the expression of the mutant were carried out according to Example 2 and Example 3. The amino acid sequence of the LbCas12a enzyme mutant obtained in Comparative Example 1 and the nucleotide gene sequence encoding the mutant are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively:

[0051]

[0052]

[0053] The mutant K20F was reacted at 37℃, 46℃, 48℃, 50℃, 52℃, 54℃ and 56℃ respectively to detect its enzyme activity at different temperatures. Figure 6 As shown, the mutant K20F has enzyme activity at 37°C, but no cleavage activity at 46-56°C.

[0054] Comparative Example 2, enzyme activity detection of original LbCas12a enzyme at different temperatures

[0055] The amino acid sequence of the original LbCas12a enzyme without mutation and the nucleotide gene sequence encoding the original LbCas12a enzyme are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively:

[0056]

[0057]

[0058] The original LbCas12a enzyme was reacted at 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, and 48°C, and its enzyme activity at different temperatures was detected. Figure 7 As shown, the reaction tube that emits fluorescence proves that the LbCas12a enzyme has enzymatic activity at this temperature. The experimental results show that the original enzyme has good enzymatic activity at 37°C-40°C, and its cutting activity is poor after it is higher than 41°C.

[0059] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A LbCas12a artificially modified enzyme with improved thermal stability, characterized in that The amino acid sequence of the LbCas12a artificially modified enzyme is shown in SEQ ID NO.

1.

2. A LbCas12a artificially modified enzyme nucleotide gene, characterized in that, The nucleotide gene encodes the thermostable LbCas12a artificially modified enzyme according to claim 1, and the nucleotide gene sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the LbCas12a artificially modified enzyme nucleotide gene according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is pET-28a (+).

5. A host cell, characterized in that The host cell comprises the recombinant expression vector according to claim 3 or 4.

6. The host cell according to claim 5, characterized in that The host cell is Escherichia coli.

7. The host cell according to claim 6, characterized in that The Escherichia coli is Escherichia coli BL21.

8. The application of the thermostable LbCas12a artificial modification enzyme according to claim 1 in the CRISPR / Cas12a system reaction, wherein the application is for non-disease diagnosis or treatment purposes.

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