Application of a new editing tool CeCas12a-A169R-F843L in gene editing

Abstract

The present invention discloses a novel editing tool CeCas12a‑A169R‑F843L application in gene editing, belonging to the field of biomedicine. The CeCas12a‑A169R‑F843L is a mutant of CeCas12a, and its amino acid sequence is shown in SED IDNO.1. The present invention is first modified by protein, so that the gene editing efficiency of variant CeCas12a‑A169R‑F843L is higher than that of wild-type CeCas12a, so as to be more conducive to the application of gene editing; The CeCas12a‑A169R‑F843L can be carried out gene editing on eukaryotic genomes at a fixed point under the mediation of crRNA and has higher gene editing efficiency. The emergence of CeCas12a‑A169R‑F843L further expands the gene editing toolbox, which is very important for basic scientific research and clinical treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an application of a CeCas12a variant (CeCas12a-A169R-F843L) in gene editing. Background Art

[0002] CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. It is an acquired immune system found in bacteria and archaea that helps them recognize and eliminate invading viruses through a specialized protease.

[0003] The most pivotal event in the history of CRISPR / Cas technology was the discovery in August 2012 by American biologist Jennifer Doudna and French biologist Emmanuelle Charpentier of CRISPR / Cas9, a dual-RNA-guided DNA endonuclease. This marked the first time that the bacterial innate immune system was repurposed as a CRISPR / Cas9 gene-editing tool. In January 2013, Feng Zhang and George Church's teams independently successfully demonstrated CRISPR gene editing in mammalian cells. Subsequently, in 2015, Feng Zhang's team reported the discovery of 16 Cas12a strains from various strains. Among them, CRISPR / AsCas12a and CRISPR / LbCas12a were capable of gene editing. Subsequent studies also reported that CRISPR / FnCas12a, CRISPR / MbCas12a, and CRISPR / CeCas12a also possessed efficient gene editing capabilities. Furthermore, CeCas12a exhibited a lower risk of off-target effects than other Cas12a strains.

[0004] Although both the CRISPR / Cas9 system and the CRISPR / Cas12a system belong to the second type of CRISPR / Cas system, and both are nucleases that perform their gene editing functions through the guidance of gRNA (guide RNA), there are certain differences between the two systems. (1) The crRNA processing and maturation process of the two is different. The pre-crRNA of Cas9 requires the help of tracrRNA and RNase III to be processed and matured, while Cas12a uses its own RNase activity to directly process the pre-crRNA. (2) The gRNAs of the two are different. The gRNA of Cas9 is a crRNA:tracrRNA complex or an sgRNA formed by the fusion of these two crRNAs, while the gRNA of Cas12a is crRNA. In general, the gRNA of Cas12a is smaller. (3) The PAM sequences recognized by the two are different. Cas9 generally recognizes a guanine-rich sequence (such as NGG) at the 3′ end of the pre-spacer sequence, while Cas12a generally recognizes a thymine-rich sequence (such as TTTN) at the 5′ end of the pre-spacer sequence. (4) The cutting methods of the two are different. The cleavage domain of Cas9 is composed of the HNH domain and the RuvC domain, which are responsible for the cleavage of the target chain and the non-target chain respectively, and the cleavage of double-stranded DNA produces a flat-ended gap. The cleavage domain of Cas12a is composed of the RuvC domain and the NuC domain, and the two domains together form a cleavage pocket that cleaves the non-target chain and the target chain in turn, and the cleavage of double-stranded DNA produces a sticky-ended gap. (5) Cas12a also has trans-cleavage activity. After it cleaves the DNA chain, its trans-cleavage activity is activated, so that it can cut any single-stranded DNA, while Cas9 does not have this property.

[0005] Therefore, as a new gene editing tool, Cas12a provides a powerful tool for scientific research and disease treatment, especially in the context of COVID-19. Based on the existing research on Cas12a, it is of great significance to discover more Cas12a variants with certain characteristics in order to respond to gene editing events in various situations in the future. Summary of the Invention

[0006] The object of the present invention is to provide an application of a CeCas12a variant (CeCas12a-A169R-F843L) in gene editing in response to the deficiencies in the prior art, so that gene editing efficiency is higher and the application range is wider.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0008] The present invention obtains a CeCas12a variant with higher gene editing efficiency by modifying the wild-type CeCas12a ("Class II V CRISPR protein CeCas12a and its application in gene editing", application number 201911119880.3).

[0009] A CeCas12a variant protein is CeCas12a-A169R-F843L, and its amino acid sequence is shown in SED ID NO.1.

[0010] The PAM sequence recognized by the above CeCas12a-A169R-F843L is a T-rich sequence, such as TTTA, TTTC, and TTTG.

[0011] The above-mentioned CeCas12a-A169R-F843L has higher gene editing efficiency in vivo than the corresponding wild-type CeCas12a.

[0012] The above-mentioned application of CeCas12a-A169R-F843L in gene editing includes prokaryotic gene editing, eukaryotic gene editing, and in vitro gene editing.

[0013] The above-mentioned application of CeCas12a-A169R-F843L in DNA detection.

[0014] The amino acid sequence of the protein CeCas12a-A169R-F843L of the present invention is as follows:

[0015] CeCas12a-A169R-F843L(SED ID NO.1):NNNTNNSFEPFIGGNSVSSKTLRNELRVGSE YTGKHIKECAIIAEDAVKAENQYIVKEMMDDFYRDFINRKLDALQGINWEQLFDIMKKAKLDKSNKVSKELDKIQESTRKEIVKIFSSDPIYKDMLKADMISKILPEYIVDKYGDAASRIEAVKVFYGFSGYFIDFW RSRKNVFSDKNIASAIPHRIVNVNARIHLDNITAFNRIAEIAGDEVAGIAEDACAYLQNMSLEDVFTGACYGEFICQKDIDRYNNICGVINQHMNQYCQNKKISRSKFKMERLHKQILCRSESGFEIPIGFQTDGEVIDAINSFSTILEEKDILDRLRTLSQEVTGYDMERIYVSSKAFESVSKYIDHKWDVIASSMYNYFSGAVRGKDDKKDAKIQTEIKKIKSCSLLDLKKLVDMYYKMDGMCLEHEATEYVAGITEILVDFNYKTFDMDDSVKMIQNEHMINEIKEYLDTYMSIYHWAKDFMIDELVDRDMEFYSELDEIYYDLSDIVPLYNKVRNYVTQKPYSQDKIKLNFGSPTLANGWSKSKEFDNNVVVLLRDEKIYLAILNVGNKPSKDIMAGEDRRRSDTDYKKMNYYLLPGASKTLPHVFISSNAWKKSHGIPDEIMYGYNQNKHLKSSPNFDLEFCRKLIDYYKECIDSYPNYQIFNFKFAATETYNDISEFYKDVERQGYKIEWSYISEDDINQMDRDGQIYLFQIYNKDFAPNSKGMQNLHTLYLKNIFSEENLSDVVIKLNGEAELFFRKSSIQHKRGHKKGSVLVNKTYKTTEKTENGQGEIEVIESVPDQCYLELVKYWSEGGVGQLSEEASKYKDKVSHYAATMDIVKDRRYTEDKF LIHMPITINFKADNRNNVNEKVLKFIAENDDLHVIGIDRGERNLLYVSVIDSRGRIVEQKSFNIVENYESSKNVIRRHDYKGKLVNKEHYRNEARKSWKEIGKIKEIKEGYL SQVIHEISKLVLKYNAIIVMEDLNYGFKRGRFKVERQVYQKFETMLINKLAYLVDKSRAVDEPGGLLKGYQLTYVPDNLGELGSQCGIIFYVPAAYTSKIDPVTGFVDVFD FKAYSNAEARLDFINKLDCIRYDASRNKFEIAFDYGNFRTHHTTLAKTSWTIFIHGDRIKKERGSYGWKDEIIDIEARIRKLFEDTDIEYADGHNLIGDINELESPIQKKF VGELFDIIRFTVQLRNSKSEKYDGTEKEYDKIISPVMDEEGVFFTTDSYIRADGTELPKDADANGAYCIALKGLYDVLAVKKYWKEGEKFDRKLLAITNYNWFDFIQNRRF.

[0016] Beneficial effects of the present invention: The present invention designed for the first time a CeCas12a variant CeCas12a-A169R-F843L with higher gene editing efficiency. This variant has higher gene editing efficiency in vivo than the wild-type CeCas12a, providing an important alternative tool for subsequent gene editing in various different situations, and plays a very important role in basic scientific research and clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the experimental flow chart of Example 1.

[0018] Figure 2 This is the agarose nucleic acid gel electrophoresis diagram after gene editing of CeCas12a-A169R-F843L in HEK293T cells.

[0019] Figure 3 This is a statistical chart of gene editing efficiency after gene editing experiments in HEK293T cells. DETAILED DESCRIPTION

[0020] In order to better understand the present invention, the content of the present invention is further illustrated below in conjunction with the examples, but the content of the present invention is not limited to the following examples.

[0021] The plasmids used in the following examples were constructed as follows:

[0022]

[0023] CeCas12a-A169R-F843L expression plasmid pcDNA3.1-CeCas12a-A169R-F843L: Using pcDNA3.1-CeCas12a plasmid as a template, A169R and F843L mutations were introduced by upstream primer F (Ce-A169R): CGGCTACTTCATCGACTTCTGGcGCAGCCGCAAGAACGTGTTCA and downstream primer R (Ce-F843L): GTTGATGGTGATGGGCATGTGGATGAgGAACTTGTCCTCGGTGTAGCGGCGG to construct pcDNA3.1-CeCas12a-A169R-F843L.

[0024] pU6-crRNA plasmid: pU6-As-crRNA (Addgene plasmids#78956) was used as the cloning vector, and the NotI and XbaI restriction sites were selected to obtain the linearized vector. At the same time, the plasmid pU6-As-crRNA was used as the template, and the upstream primer NotI-U6-F: cacgagactagcctcgagcggccgcccccttcaccgagggcctatttcccatg and the corresponding downstream primers (including B2M-crRNA-R: cccgtacatcgcgaatctagaaaaaaCATTCTCTGCTGGATGACGTGAGatctacaagagtagaaattacg, site5-crRNA-R: cccgtacatcgcgaatctagaaaaaaGTAACAGGTATGGACCATCAatctacaagagtag aaattacg, DNMT1-1-R :cccgtacatcgcgaatctagaaaaaaGAGTAACAGACATGGACCATCAGatctacaagagtagaaattacg, TAX1BP3-crRNA-R: gcccgtacatcgcgaatctagaaaaaataaaaGTTTCTGAATGGCCTATGTGatctacaagagtagaaatt, NLRC4-crRNA-R :gcccgtacatcgcgaatctagaaaaaaataaaaTATCAACTTGTGTCTCCCTCatctacaagagtagaaatt, CLIC4-crRNA-R :gcccgtacatcgcgaatctagaaaaaaataaaaGGTAGGGGAGGTAGCCAGGGatctacaagagtagaaatt) was obtained by PCR to obtain an insert fragment containing two restriction sites, NotⅠ and XbaⅠ, and then the complete pU6-B2M-crRNA, pU6-site5-crRNA, pU6-DNMT1-1-crRNA, pU6-TAX1BP3-crRNA, pU6-NLRC4-crRNA, and pU6-CLIC4-crRNA plasmids were obtained by recombination reaction.

[0025] Example 1

[0026] Six HEK293T cell endogenous gene targets, including β2M, HEK293T site5, DNMT1-1, TAX1BP3, NLRC4, and CLIC4, were selected. Figure 1 The experiment was carried out according to the experimental procedure shown.

[0027] (1) HEK293T cells were transfected with CeCas12a expression plasmid pcDNA3.1-CeCas12a or its variant CeCas12a-A169R-F843L expression plasmid pcDNA3.1-CeCas12a-A169R-F843L (600ng) and corresponding pU6-crRNA plasmid (300ng). 50μL plasmid mixture was mixed with 50μL transfection reagent mixture and incubated for 20 minutes before transfection. Before transfection, 1mL basal culture medium (DMEM, purchased from Gibco) was added to each well of a 24-well plate in which HEK293T cells were cultured, and 1mL culture medium was aspirated. After 4 hours and 30 minutes of transfection, 350μL culture medium was aspirated from each well and 350μL complete culture medium (DMEM supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin) was carefully added. The genome was extracted after 48 hours.

[0028] (2) The cells in the 24-well plate were pipetted evenly, transferred to a centrifuge tube, and centrifuged at 4000 g for 2 minutes. The supernatant was discarded, and 50 μL of lysis buffer and 0.5 μL of protease were added and pipetted evenly. After incubation at 55°C for 30 minutes and 95°C for 20 minutes, 1 μL of supernatant was used as a template for PCR. The PCR primer sequences are listed in Table 1. The amplified product was verified as a single band by agarose gel electrophoresis, and the PCR product was purified and the concentration was determined.

[0029] Table 1 Gene targets and target amplification primer sequences

[0030]

[0031]

[0032] (3) Take 250 ng of PCR product and perform in vitro annealing directly. Add 1 μL of 10×T7 buffer and 250 ng of amplified fragment to a PCR tube and fill with sterile water to 10 μL. Place the prepared system in a PCR instrument and set the annealing program to 95℃ for 3 minutes, 95℃ for 30 seconds, 90℃ for 30 seconds, 85℃ for 30 seconds, 80℃ for 30 seconds, 75℃ for 30 seconds, 70℃ for 30 seconds, 65℃ for 30 seconds, 60℃ for 30 seconds, 55℃ for 30 seconds, 50℃ for 30 seconds, 45℃ for 30 seconds, 40℃ for 30 seconds, 35℃ for 30 seconds, 30℃ for 30 seconds, and 25℃ for 1 minute.

[0033] (4) Prepare a T7E1 enzyme digestion reaction system solution (0.2 μL 100× T7 buffer, 0.25 μL T7E1 enzyme, 1.55 μL water), add the solution to the renaturation reaction system, and mix well. After reacting at 37°C for 20 minutes, perform 2% agarose gel electrophoresis to determine the digestion status and perform grayscale analysis on the electrophoresis bands. The results are as follows: Figure 2 As shown, both wild-type CeCas12a and CeCas12a-A169R-F843L showed cleavage bands at the six target sites of β2M, HEK293T site5, DNMT1-1, TAX1BP3, NLRC4, and CLIC4.

[0034] The above experiment was repeated three times, and the cutting efficiency statistics were obtained by counting the ratio of the cutting sub-band and the amplified main band, that is, Figure 3 The results showed that the variant CeCas12a-A169R-F843L was 0.6 to 3.2 times more efficient than the wild type in editing six endogenous gene targets. (The six genes in this example are only used as representative examples).

Claims

1. A CeCas12a variant CeCas12a-A169R-F843L, characterised in that The amino acid sequence of the CeCas12a-A169R-F843L is shown in SED ID NO.

1.

2. CeCas12a variant CeCas12a-A169R-F843L according to claim 1, is characterized in that: The PAM sequence recognized by the CeCas12a-A169R-F843L is a T-rich sequence.

3. CeCas12a variant CeCas12a-A169R-F843L according to claim 2, is characterized in that: The PAM sequences include TTTA, TTTC, and TTTG.

4. The application of the CeCas12a variant CeCas12a-A169R-F843L described in claim 1 in gene editing, characterized in that: The gene editing is in vitro gene editing.

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