CN116376945B

CN116376945B - I-type caspase gene insertion tool and application

Info

Publication number: CN116376945B
Application number: CN202310318216.1A
Authority: CN
Inventors: 肖易倍; 陆美玲; 尹捷; 任科静
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2023-03-28
Filing date: 2023-03-28
Publication date: 2024-01-23
Anticipated expiration: 2043-03-28
Also published as: CN116376945A

Abstract

The invention relates to a type I caspase gene insertion tool and application thereof. The I-type caspase gene inserting tool consists of caspase protein, mini-caspaseon containing target gene segment and target-ccdB carrier. The application is editing prokaryotic or eukaryotic genes. According to the invention, caspase protein, mini-Caspo containing specific terminal inversion repeated sequences (TIRF and TIRR) and target-ccdB vector are utilized to identify and insert target site repeated sequences (TSDs) in a targeting manner, so that the target gene fragment fixing target fixing direction is inserted, the insertion of a long fragment gene can be realized, and the exposed DNA double strand is ensured not to break in the editing process, thereby being more efficient and safer.

Description

I-type caspase gene insertion tool and application

Technical Field

The invention relates to an I-type caspase gene insertion tool and application thereof, belonging to the technical field of biological medicine.

Background

Casnoson is a new class of Mobile Genetic Elements (MGEs) found in certain bacteria and archaebacteria that are associated with a set of variable transposon-related genes, such as type B DNA polymerase (Poly B). These clusters are flanked by inverted terminal repeats (TIRs), surrounded by direct repeats known as target site repeats (TSDs), and the like ^[1] . However, unlike the transposon system, there is no transposase that integrates MGEs into the host chromosome in the gene encoded by caspase, but there is a "Cas1" gene homologous to CRISPR-Cas1, and this specific Cas1 encoded by Cas1 is caspase ^[1,2,3] . And (3) withTypical cas1 genes from the CRISPR-cas system differ, although cas 4-like genes have been identified in some casbasses, casbasses are independent of CRISPR sites or other cas genes.

Currently, casnoson can be divided into four families based on comparative genomics, phylogenetic and taxonomic distribution analysis of Cas1. Wherein almost all caspase studies at this stage have focused on type 2 family A. Boost and M. Mazei caspases, while other families of caspase studies have been limited to bioinformatics ^[4,5] . Compared with type II caspases, the C-terminal of type I caspases naturally lacks the HTH domain, making its molecular weight smaller and more chemically similar to CRISPR-Cas1. Currently, there is document verification ^[6] Ca.n.koreensis caspases of type I family also catalyze the full integration of target sequences.

Unlike CRISPR-Cas systems, which require both Cas1 and Cas2 proteins to complete integration in vitro, for caspase-catalyzed integration reactions, the integration reaction can be completed by means of only caspases homologous to CRISPR-Cas1. Under the action of caspase, dsTIR sequences can specifically attack target sequences consisting of leader and TSD sequences, integration usually occurs at both sides of the TSD sequence, i.e., at both the leader and the spacer ends of the TSD (as shown in FIG. 1).

From an processive point of view, this transposon-like integration function results from the polB-like gene in the caspase gene. A transposon is a DNA fragment that is capable of changing its position in the genome. The most prominent mechanism of transposon movement is the "cut-and-paste" transposon, during which the transposon enzyme mediates excision of elements from their donor sites and re-integration into new chromosomal sites (as shown in FIG. 2). During transposition, the transposon (i) interacts with binding sites in Terminal Inverted Repeats (TIRs) that border the transposon, (ii) facilitates assembly of synaptic complexes (also known as paired-end complexes (PECs)), (iii) catalyzes excision of the element from its donor site, and (iv) integrates the excised transposon into a new location of the target DNA ^[7,8] All of these processes are for Mg ²⁺ Ion dependence ^[9,10] . And caspase-catalyzed integration reactions in theseAll exhibited high similarity to transposon systems in character.

Currently, transposon systems developed based on the polB gene, such as Sleeping Beauty, piggyBac and Tol2, have been matured. However, these transposon kits are between 1k and 5k in length ^[11] As shown in FIG. 3, the transposon is too large for use, and several elements are added after TIR, which also makes the two sides of the constructed target gene carry unavoidable redundant elements; the screening mode is generally simple resistance screening, and the target gene itself is required to carry different resistance genes during construction, so that the method is inconvenient to use.

A series of CRISPR tools, represented by the CRISPR-Cas9 system, are currently simple and easy to operate, but precise typing of exogenous DNA at targeted double strand breaks remains difficult. Successful precise knock-in of exogenous DNA requires both efficient cleavage by targeting endonucleases and recruitment of endogenous DNA repair factors to incorporate the desired editing into the host genome. However, the CRISPR-Cas9 system itself cannot meet the requirement of accurate typing, and exogenous functional factors need to be introduced to achieve this function. Canny et al ^[12] Jayavanadaham et al ^[13] The idea of inhibiting non-homologous end joining by promoting protein 53BP1 was presented in 2018 and 2019 sequentially. Charpentier ^[14] ，Nakade ^[15] ，Tran ^[16] The inventors believe that other methods that promote end excision are crucial for improving the efficiency of homologous recombination. In addition to this, rees et al ^[17] Selecting a function of participating in DNA damage repair by fusing RAD51 and Cas9 and utilizing a strand-exchange factor PAD51 to directly target a homologous recombination mechanism so as to enhance the efficiency of precisely knocking in exogenous DNA by a CRISPR-Cas9 system. Eghbalsaied et al ^[18] Then, it is proposed to pretreat different types of cells with the antitumor drug Nocodazole, and to stop the cells in the G2/M phase of the cell cycle by interfering with microtubule polymerization, and simultaneously introduce RNase HII to enhance the treatment of R-loop and the excision repair of ribonucleotides by eukaryotic cells to enhance the homology directed repair of single-stranded and double-stranded DNA. However, at present, the endogenous factors directly influencing homologous recombination are not known, and the mechanism and the direct influence on homologous recombination are not knownClearly, the introduction of exogenous factors that have an impact on the cell cycle also presents new problems for subsequent in vivo applications, and thus the development of CRISPR-Cas9 systems for precise gene insertion is still underway. And even if CRISPR-Cas9 cuts target DNA, some target proteins can be partially expressed due to exon skipping or translation reinitiation mechanism, and activity is generated to play a role, so that the efficiency of gene insertion is affected ^[19] . Meanwhile, CRISPR-Cas9 is relatively limited in application of long fragment insertion, and also limited in application ^[20] . And the CRISPR system inevitably leads to double-stranded DNA break in the editing process, and the non-conservative non-homologous end joining (NHEJ) repair route caused by double-stranded DNA break can bring unpredictable error repair and mismatch in the practical application process, and uncontrollable byproducts are easy to generate, so that the gene insertion tool has potential safety hazard.

Compared with the CRISPR system and the transposon system, the TIR length of the type I caspase system is only 20nt, the length of the targeting sequence where the integration site TSD is positioned is only 35bp, and the whole system is less than 1% of the transposon element, so that the type I caspase system can be easily compatible with the existing commonly used prokaryotic expression tool vectors and eukaryotic expression tool vectors. In the result of caspase-mediated integration, only 6 amino acids are added in front of the target gene, and the subsequent expression or activity experiment is not affected. And unlike CRISPR system which can cause double-strand DNA break in the process of gene editing, the target gene containing TIR in the process of caspase mediated integration can not break, which greatly reduces the possibility of DNA mismatch and repair error, and unlike CRISPR-Cas9 system which lacks long fragment integration function, caspase system can integrate target gene with 100bp-7000bp size. Therefore, the inventors' subject group studied that the Class 1CRISPR-Cas system is expected to be a better and more powerful gene insertion tool, and is worthy of research to obtain a new gene insertion tool.

The references referred to above are as follows:

[1].Krupovic M,Beguin P,Koonin EV,et al.Casposons:mobile genetic elements that gave rise to the CRISPR-Cas adaptation machinery[J].Current Opinion in Microbiology 2017,38:36-43.

[2].Koonin EV,Makarova KS,et al.Mobile Genetic Elements and Evolution of CRISPR-Cas Systems:All the Way There and Back[J].Genome Biol Evol 2017,9(10):2812-2825.

[3].Makarova KS,Wolf YI,Shmakov SA,et al.Unprecedented Diversity of Unique CRISPR-Cas-Related Systems and Cas1 Homologs in Asgard Archaea[J].CRISPR J 2020,3(3):156-163.

[4].Hickman AB,Kailasan S,Genzor P,et al.Casposase structure and the mechanistic link between DNAtransposition and spacer acquisition by CRISPR-Cas[J].Elife 2020,9.

[5].Koonin EV,Makarova KS,et al.Origins and evolution of CRISPR-Cas systems[J].Philos Trans R Soc Lond B Biol Sci 2019,374(1772):20180087.

[6].Wang X,Lu M,Xiao Y,et al.Sequence specific integration by the family 1casposase from Candidatus Nitroopumilus koreensis AR1[J].Nucleic Acids Res.2021Sep 27；49(17):9938-9952.

[7].Kulkosky J,Jones,et al.Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases[J].Mol.Cell.Biol.1992,12,2331–2338.

[8].Doak T.G,Doerder F.P,et al.A proposed superfamily of transposase genes:Transposon-like elements in ciliated protozoa and a common“D35E”motif[J].Proc.Natl.Acad.Sci.USA 1994,91,942–946.

[9].Bujacz G,Alexandratos J,et al.Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity[J].J.Biol.Chem.1997,272,18161–18168.

[10].Goldgur Y,Dyda F,et al.Three new structures of the core domain of HIV-1integrase:An active site that binds magnesium[J].Proc.Natl.Acad.Sci.USA 1998,95,9150–9154.

[11].Sandoval-Villegas N,Nurieva W,et al.Contemporary Transposon Tools:A Review and Guide through Mechanisms and Applications of Sleeping Beauty,piggyBac and Tol2 for Genome Engineering[J].Int J Mol Sci.2021May 11；22(10):5084.

[12].Canny M.D,Moatti N,et al.Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency[J].Nat.Biotechnol.2018,36,95–102.

[13].Jayavaradhan R,Pillis M,et al.CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites[J].Nat.Commun.2019,10,2866.

[14].Charpentier M,Khedher Y,et al.CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair[J].Nat.Commun.2018,9,1133.

[15].Nakade S,Mochida K,et al.Biased genome editing using the local accumulation of DSB repair molecules system[J].Nat.Commun.2018,9,3270.

[16].Tran T,Bashir S,et al.Enhancement of Precise Gene Editing by the Association of Cas9 With Homologous Recombination Factors[J].Front.Genet.2019,10,365.

[17].Rees A,Yeh H,et al.Development of hRad51-Cas9 nickase fusions that mediate HDR without double-stranded breaks[J].Nat.Commun.2019,10,2212.

[18].Eghbalsaied S,Kues W,et al.CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII[J].Sci Rep.2023Feb 15；13(1):2690.

[19].Xiao Y,Luo M,et al.Structure basis for RNA-guided DNA degradation by Cascade and Cas3[J].Science,2018,361(6397).

[20].Smits A.H,Ziebell F,et al.Biological plasticity rescues target activity in CRISPR knock outs[J],Nat Methods 16(11)(2019)1087-1093.

disclosure of Invention

The main purpose of the invention is as follows: the I-type caspase gene insertion tool can overcome the defect of DNA double-strand break in the current CRISPR gene insertion process, reduce the risk of double-strand break introduction in the in-vivo editing process, and is more efficient and safer. Also provides the application of the system.

The technical scheme for solving the technical problems is as follows:

a type I caspase gene insertion tool is characterized by comprising a caspase protein, mini-caspaseon containing target gene fragments and target-ccdB vector; the amino acid sequence of the caspase protein is SEQ ID NO.2; the Mini-Caspo is dsDNA, and has the structure that one end of a target gene fragment is connected with a TIRF sequence, the other end of the target gene fragment is connected with a TIRR sequence, the nucleotide sequence of the TIRF sequence is SEQ ID NO.6, and the nucleotide sequence of the TIRR sequence is SEQ ID NO.7; the target-ccdB vector contains a target gene fragment and a coding gene fragment of ccdB protein, the sequence of the target gene fragment is SEQ ID NO.3, the amino acid sequence of the ccdB protein is SEQ ID NO.5, and the coding gene fragment of the ccdB protein corresponds to the amino acid sequence of the ccdB protein.

The system utilizes caspase protein, mini-caspaseon containing specific terminal inversion repeated sequences (TIRF, TIRR) and target-ccdB carrier to identify and insert target site repeated sequences (TSDs) in a targeting manner, so that the target gene fragment fixing target fixing direction is inserted more strictly; the insertion of the long fragment gene can be realized, and the DNA double strand exposed outside can be ensured not to break in the editing process, so that unpredictable off-target and mutation caused by broken gene editing can be avoided.

Preferably, the Mini-Caspo structure is a TIRF sequence, a target gene fragment and a TIRR sequence from upstream to downstream; the target-ccdB vector contains the lac UV5 promoter. Thus, the specific structure of Mini-Caspo can be further optimized, and the promoter of the target-ccdB vector can be optimized.

Preferably, the coding gene sequence of the caspase protein is SEQ ID NO.1; the coding gene fragment sequence of the ccdB protein is SEQ ID NO.4. Thus, the coding gene sequence of the caspase protein and the ccdB protein can be further optimized.

Preferably, the caspase protein and target gene fragment are derived from ca.n.koreens, respectively. This further optimizes the specific source of caspase protein, target gene fragment.

By adopting the preferable scheme, the specific detail technical characteristics can be further optimized, so that a better gene insertion effect is realized.

The invention also proposes:

a gene insertion method is characterized in that the I-type caspase gene insertion tool is adopted;

the gene insertion method comprises the following steps:

firstly, preparing caspase protein; preparing Mini-Caspo containing target gene fragment; constructing a target-ccdB carrier;

secondly, uniformly mixing caspase protein and Mini-Casposon, target-ccdB carrier for reaction to catalyze the integration of Mini-Caspo, and recovering an integrated product after the reaction is stopped;

thirdly, after the integration product is electrically transferred to the sensitive cells, the integration product is cultured by adopting a resistance culture medium containing antibiotics, and the grown monoclonal is collected to obtain the gene integration product containing the target gene fragment.

The method utilizes caspase protein of the system, mini-Caspo containing specific terminal inversion repetitive sequences (TIRF, TIRR) and target-ccdB carrier to identify and insert target site repetitive sequences (TSDs) in a targeting way, so as to realize the fixed-direction insertion of target spots of target gene fragments; not only can realize the insertion of long fragment genes, but also can ensure that DNA double chains cannot break in the editing process so as to avoid unpredictable off-target and mutation caused by broken gene editing.

Preferably, in the first step, the caspase protein is prepared by the following steps:

s1, constructing a plasmid containing a coding gene sequence of caspase protein according to the amino acid sequence of the caspase protein, transferring the plasmid into competent cells by a chemical conversion method for culture, extracting the plasmid, and obtaining a correct recombinant plasmid by Sanger sequencing;

s2, performing induction culture on strains containing the plasmids, and purifying by crushing thalli, centrifuging and nickel column affinity chromatography to obtain caspase protein. The specific preparation process of the caspase protein can be further optimized.

Preferably, in the first step, the Mini-casoson containing the target gene fragment is prepared by the following steps:

designing an upstream amplification primer and a downstream amplification primer according to a target gene fragment, adding a TIRF sequence at the 5 'end of the upstream amplification primer, adding a TIRR sequence at the 5' end of the downstream amplification primer to obtain a specific primer, amplifying Mini-Caspo with a structure of the TIRF sequence-target gene fragment-TIRR sequence by using the specific primer through a PCR (polymerase chain reaction) mode, and finally obtaining purified Mini-Caspo by a gel cutting recovery mode. Thus, the specific preparation process of Mini-Caspo can be further optimized.

Preferably, in the first step, the target-ccdB vector is pET28a-lac UV5 master-Ca.N.koreensis target-ccdB; in S1, the plasmid containing the coding gene sequence of the caspase protein is pET-28a-ts-sumo-Ca.N. koreensis-caspase. This further optimizes the target-ccdB vector, and the plasmid containing the gene sequence encoding the caspase protein.

Preferably, in the second step, in the reaction system, the final concentration of caspase protein is 1+ -0.1 μM (i.e. μmol/L, the same applies hereinafter), the final concentration of Mini-Caspo is 0.2+ -0.05 μM, and the final concentration of target-ccdB carrier is 65+ -5 ng/. Mu.L; the reaction conditions are that the reaction is carried out for at least 60min at 37+/-0.5 ℃ under the condition of 1X Integration buffer; EDTA was added at the termination of the reaction to terminate the reaction; and (5) recovering the integrated product by using a PCR product recovery kit. The specific reaction process and reaction parameters of the second step can be further optimized in this way.

In the third step, the competent cell is DH10B; when the electric power is turned, an electric power turning instrument is adopted, and electric shock parameters are as follows: 2.5kV/cm, resistance 200 omega, capacitance 25 muF, time 5ms; after electrotransformation, culturing in SOC non-antibiotic liquid culture medium, and then coating bacterial liquid in kanamycin sulfate-containing resistance plate for culturing, wherein the normal monoclonal is the gene integration product containing target gene fragment. The specific reaction process and reaction parameters of the third step can be further optimized.

The invention also proposes:

the use of the above-described caspase I gene insert tool for editing prokaryotic or eukaryotic genes.

Compared with the prior art, the I-type caspase gene insertion tool provided by the invention utilizes caspase protein, mini-caspase containing specific terminal inversion repetitive sequences (TIRF and TIRR) and target-ccdB vector to identify and insert target site repetitive sequences (TSDs) in a targeting manner, realizes the fixation direction insertion of a target gene fragment fixation target, can realize the insertion of a long fragment gene, ensures that DNA double chains exposed outside cannot break in the editing process, and is more efficient and safe.

Drawings

FIG. 1 is a diagram showing the comparison of CRISPR system integration mechanism with type I caspase system integration mechanism in the background of the invention.

FIG. 2 is a schematic diagram showing an overview of transposase-mediated cut-and-paste transposition in the background of the invention.

FIG. 3 is a schematic diagram of the organization and functional domains of autonomous transposable elements and transposases of Sleep Beauty (SB), piggyBac (PB) and Tol2 in the background of the invention.

FIG. 4 shows a plasmid map of pET28a-TS-SUMO-Ca.N.koreensis caspase constructed according to example 1 of the present invention.

FIG. 5 is a schematic representation of the expression and purification results of Ca.N.koreensis caspase protein according to example 2 of the present invention.

FIG. 6 shows a plasmid map of PUC19-Ca.N.koreensis-target constructed in example 3 of the present invention.

FIG. 7 is a diagram showing the total integration of Mini-casinos in example 3 of the present invention.

FIG. 8 is a plasmid map of pET28a-lac UV5promoter-Ca.N.koreensis target-ccdB in example 4 of the present invention.

FIG. 9 is a graph showing the determination of the TIR key sites in Ca.N.koreensis koreensis casposase integration in example 4 of the present invention.

FIG. 10 is a schematic diagram of a modified Mini-Casboost in example 4 of the present invention.

FIG. 11 is a plasmid map of pLtDuet-p3c-Cas2Cas3 constructed in example 5 of the present invention.

FIG. 12 is a graph showing the results of Sanger sequencing after 7 k-pltDuet-Mini-Casboost total integration in example 5 of the present invention.

Detailed Description

The invention is described in further detail below with reference to the accompanying drawings in combination with embodiments. The invention is not limited to the examples given.

Example 1

This example is a construction of pET28a-TS-SUMO-Ca.N. koreensis-caspase plasmid for subsequent gene insertion.

Amplifying a Ca.N.koreensis-caspase sequence (SEQ ID NO. 1) by using PCR, obtaining a recombinant plasmid by enzyme digestion and enzyme ligation by using a vector pET28a, and transferring the recombinant plasmid into DH5 alpha competence by using a chemical conversion method; the plasmid was then extracted using plasmid extraction Kit and the correct recombinant plasmid was obtained after Sanger sequencing.

The construction of pET28a-TS-SUMO-Ca.N. koreensis-caspase plasmid structure by the above-described method is shown in FIG. 4.

The sequences related to the above are as follows:

coding gene sequence of caspase protein: SEQ ID NO.1:

atgaccctgaagaaccagaagaaccactacaacgtgaagtttctgaaaggttatggccacagcattagcgttaaggacagcaaaatcattctgaaaagca

accacgatccgtttagcccgccggagcaggaagagtggttcgtgaagaacatgccgtacgaaaaaattgtgctgagcggcaagggctatgttagcacc

gaggcgctgagcctgctgagcgaaaacaaccgtaacgtgatcctgctggacaaccacggtaaaccggttaccttttgcaacggcatgatggacagcct

gaccgcgaccaagtaccgtatggcgcaatatgataccttccgtaacccggagaaatgcgaatacctgcgtaagcagatcattagcgcgaagaaagaga

gccaactgaaactgctgcgtctgattggtagcgagatcagcgaactgccggatagcgaacacattagcgcgaaggtgtactggaccgagttcgcgaag

tttatcccggaaaaatatcagttccacagccgtaaccaaagccacattaccagcagcaaaaacaacgcgaccgacatcattaacgcgctgctgaactacg

gttatagcgtgctggcgggcgagatcagcaagttcgtttgcggttttggtctggacccgtacctgggtttcatgcaccgtagccacaccggctttcaaccg

ctggtttatgacatcattgaaccgtttcgttggctggttgactacaccgtttatagcatggcgaaccacagcagcacccgtcagcgtattaagctgaaagagt

acagcttcaccaaagacggtaccgtggttctggaatatagcctgatcaagcgtttcctggagatgctggaacgtcagtttagccaagagcgtaaatacagc

ttccgtcacggtaagaaaaccaaggatggcctgaaaagcgtgcaggaaatcaccgtggttaagatcattatccaaaacctggttgagtatagcaccggca

agcagaaaagcctggaaagcaacaacttcgcgttctttgactttta

amino acid sequence of caspase protein: SEQ ID NO.2:

MTLKNQKNHYNVKFLKGYGHSISVKDSKIILKSNHDPFSPPEQEEWFVKNMPYEKIVLSGKGYV

STEALSLLSENNRNVILLDNHGKPVTFCNGMMDSLTATKYRMAQYDTFRNPEKCEYLRKQIISAK

KESQLKLLRLIGSEISELPDSEHISAKVYWTEFAKFIPEKYQFHSRNQSHITSSKNNATDIINALLNY

GYSVLAGEISKFVCGFGLDPYLGFMHRSHTGFQPLVYDIIEPFRWLVDYTVYSMANHSSTRQRIK

LKEYSFTKDGTVVLEYSLIKRFLEMLERQFSQERKYSFRHGKKTKDGLKSVQEITVVKIIIQNLVE

YSTGKQKSLESNNFAFFDF

example 2

This example is based on example 1 and the Ca.N.koreensis-caspase protein was purified.

E.coli BL21 (DE 3) strain containing pET-28 a-ts-sumo-Ca.N.koreensis-caspase plasmid was inoculated at 1% (v/v) into LB liquid medium containing 0.1% (v/v) kanamycin, cultured with shaking at 37℃at 220rpm until OD600 value was between 0.8 and 1.0, cooled in ice bath, and then added with IPTG at a final concentration of 0.5mM, and cultured at 25℃at 180rpm overnight. The overnight cultured bacteria were collected by centrifugation at 5000rpm for 10min and bacterial pellet was resuspended in Buffer A (50mM HEPES pH 7.5,10% (v/v) glycerol, 20mM imidazole and 1000mM NaCl). After the bacterial suspension is broken (800 bar,10 min) by a high-pressure bacteria breaker, the precipitate is discarded by centrifugation at 18000rpm for 30min at 4 ℃, and the supernatant containing the target protein is subjected to secondary centrifugation, and the obtained supernatant is used as a sample for nickel column affinity chromatography.

After loading onto nickel columns pre-equilibrated with Buffer A at 4℃20 column volumes of Buffer A were used to wash out non-hanging and low affinity proteins, followed by elution of 10 column volumes with Buffer B (20mM HEPES pH 7.5,4.5MNaCl) at room temperature to remove bound nucleic acids, and finally all proteins of interest were eluted with 5-10 column volumes of Buffer C (50mM HEPES pH 7.5,1M NaCl,10% (v/v) glycerol and 300mM imidazole) at 4 ℃. Adding 1% (w/w) SUMO protease into the eluate containing target protein, and cutting at 4deg.C overnight to remove SUMO tag protein. The SUMO-tagged protein can bind to the nickel column due to the 6 XHis content, while the target protein cannot bind to the nickel column due to the cleavage of the SUMO-tagged protein. The unlabeled Ca.N. korensis-caspase is concentrated by ultrafiltration concentration tube at 30K and then loaded with HiLoad 16/60Superdex 200 pre-equilibrated by Buffer D (20mM HEPES pH 7.5,150mM NaCl and 10% (v/v) glycol). And collecting peak tip parts with higher protein purity according to SDS-PAGE electrophoresis identification results of each eluted hole, concentrating to about 15mg/mL, and rapidly freezing in liquid nitrogen and storing at-80 ℃ for later use.

The corresponding results are shown in FIG. 5, wherein, A is a SDS-PAGE analysis chart of the imidazole nickel column affinity chromatography eluents with different concentrations of Ca.N. koreensis-caspase; lane 1:20mM imidazole eluate; lane 2, 50mM imidazole eluate; lane 3:300mM imidazole eluate. Panel B shows the results of gel filtration chromatography and SDS-PAGE identification of Ca.N. koreesis-caspase by HiLoad 16/600Superdex 200prep grade, in the gel filtration chromatography, the abscissa represents the elution volume, the ordinate represents the light absorption value of the corresponding wavelength, the elution volume of the peak tip of the protein is 71.51mL, the curve at the arrow is the protein absorption value change curve of A280, and the other curve is the nucleic acid absorption value change curve of A260. As can be seen, this example successfully purified Ca.N.koreensis-caspase protein using the methods described above.

Example 3

This implementation is to verify that the Ca.N.koreensis-caspase system has full integration capability.

(1) Construction of the PUC19-Ca.N.koreensis-target plasmid

Integrating Ca.N.koreensis target gene (SEQ ID NO. 3) containing specific TSD (target site repetitive sequence) into a standard PUC19 vector by a PCR mode, and transferring the recombinant plasmid into DH5 alpha competence by a chemical conversion method after homologous recombination; the correct recombinant plasmid was obtained after Sanger sequencing using plasmid extraction Kit extraction plasmid.

The structure of the PUC19-Ca.N.koreensis-target plasmid constructed by the method described above is shown in FIG. 6.

The sequence involved is as follows:

sequence of target gene: SEQ ID NO.3:

note that: in the above SEQ ID No.3, the TSD sequence is framed and the rest is the target sequence.

(2) Preparation of Mini-casspoons

The method comprises the steps of taking a KanR resistance gene as a target integration sequence, selecting fragments with different lengths of 1K-3K as target gene fragments to be inserted for amplification, adding a TIR sequence with the length of 20nt at the tail ends of original upstream and downstream amplification primers to obtain a series of primers comprising the KanR-TIR-F, kanR-TIR-1K-R, kanR-TIR-2K-R, kanR-TIR-3K-R, carrying out PCR amplification by taking the KanR resistance gene as a template, and obtaining long-chain dsDNA (KanR-Mini-Casboost) with the lengths of 1258bp, 2151bp and 3292bp containing the KanR resistance gene and the TIR sequence at two ends by cutting gel and recycling the PCR products.

The sequence involved is as follows:

TIR sequence:

tagaatctttttattccgtt

KanR-TIR-F sequence:

tagaatctttttattccgttcgctgagcaataactagcataacccc

KanR-TIR-1K-R sequence:

tagaatctttttattccgttggaagagcgctgcatgcctat

KanR-TIR-2K-R sequence:

tagaatctttttattccgttctcgaccgatgcccttgagag

KanR-TIR-3K-R sequence:

tagaatctttttattccgtttttcccgcgtggtgaaccagg

1258bp KanR-Mini-Caspo sequence:

tagaatctttttattccgttcgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaacctcaggcatttgagaa

gcacacggtcacactgcttccggtagtcaataaaccggtaaaccagcaatagacataagcggctatttaacgaccctgccctgaaccgacgacaagctg

acgaccgggtctccgcaagtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgaattaatt

cttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaact

caccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaataa

ggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagccattac

gctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcggtcgctgttaaaaggacaattacaa

acaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccg

gggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccat

ctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctgattgcc

cgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctcatactc

ttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggcatgcagcgctcttcc

aacggaataaaaagattcta

2151bp KanR-Mini-Caspo sequence:

gcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggtgtcagctcactcaaaagcggtaatacggttatccacagaatcagggg

ataaagccggaaagaacatgtgagcaaaaagcaaagcaccggaagaagccaacgccgcaggcgtttttccataggctccgcccccctgacgagcatc

acaaaaatcgacgctcaagccagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttc

cgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgttggtatctcagttcggtgtaggtcgtt

cgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgact

tatcgccactggcagcagccattggtaactgatttagaggactttgtcttgaagttatgcacctgttaaggctaaactgaaagaacagattttggtgagtgcg

gtcctccaacccacttaccttggttcaaagagttggtagctcagcgaaccttgagaaaaccaccgttggtagcggtggtttttctttatttatgagatgatgaat

caatcggtctatcaagtcaacgaacagctattccgttactctagatttcagtgcaatttatctcttcaaatgtagcacctgaagtcagccccatacgatataagt

tgtaattctcatgttagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagaacggaataaaaagattcta

3223bp KanR-Mini-Caspo sequence:

tgtaattctcatgttagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgcctaatgagtg

agctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagag

gcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttgcagcaa

gcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatcccactac

cgagatgtccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagtgggaacg

atgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtgagatattta

tgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatgctccacgc

ccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcaggcagcttc

cacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacaggcttcgacg

ccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgcagggccaga

ctggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccgcttccactttt

tcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaaaacggaataaaaagattcta

(3) Verification of the full-integration Capacity of the Ca.N-caspase System

The resulting KanR-Mini-caspases were incubated with Ca.N.koreensis caspase of example 2 and PUC19-Ca.N.koreensis-target vector of (1) above to catalyze the integration reaction of KanR-Mini-caspases. After isopropanol precipitation, the reacted product was electrotransferred to an ElectroMax E.coli DH10B competence and coated onto a double antibody plate containing kanamycin sulfate and ampicillin. Transformants grown on the double antibody plates are the products of the full integration reaction, and several monoclonal antibodies were randomly picked for Sanger sequencing. The results are shown in FIG. 7: the presence of the expected KanR-Mini-Casboost in the plasmid after the integration reaction indicated that: similar to CRISPR-Cas1, ca.n.koreensis caspase has the ability to catalyze the occurrence of fully integrated reactions.

Example 4

This example is to introduce ccdB toxic proteins and modify the original ca.n.koreensis TIR sequence to achieve targeted integration of Mini-casosons.

The Ca.N.korensis caspase full integration reaction is similar to CRISPR-Cas1, and is completed by the attack of the 3' end of the TIR sequence carried by the two ends of Mini-Casphos to the 5' end of the TSD sequence, and in the Ca.N.korensis caspase original system, different directivities can appear in the full integration process due to the fact that the sequences of the 3' ends of the TIR sequences are identical, but the random integration mode of the site-specific direction is not beneficial to the subsequent tool development. Therefore, the gene editing tool for site-specific directional fixation is developed by simultaneously modifying the integration target vector containing Ca.N.koreensis target and Ca.N.koreensis TIR, introducing the toxic protein ccdB and modifying Ca.N.koreensis TIR into an expression switch of ccdB toxic protein.

(1) Construction of pET28a-lac UV5promoter-Ca.N. koreensis target-ccdB vector

Integrating Ca.N.koreensis target gene (SEQ ID NO. 3) into a pET28a vector by utilizing homologous recombination in a PCR mode, adding ccdB protein gene (SEQ ID NO. 4) at the end of a T7 expression frame, and adding a lac UV5promoter after RBS site and reserving upstream and downstream box elements to improve the ccdB expression level because the ccdB protein is not high as phage toxic protein to be expressed on a common vector of the existing escherichia coli expression system, and transferring the recombinant plasmid into DH5 alpha competence by using a chemical conversion method after recombination; the correct recombinant plasmid was obtained after Sanger sequencing using plasmid extraction Kit extraction plasmid. The structure of pET28a-lac UV5promoter-Ca.N.koreensis target-ccdB plasmid constructed by the method described above is shown in FIG. 8.

The sequence involved is as follows:

coding gene sequence of ccdB protein: SEQ ID NO.4:

cagtttaaggtttacacctataaaagagagagccgttatcgtctgtttgtggatgtacagagtgatattattgacacgcccgggcgacggatggtgatcccc

ctggccagtgcacgtctgctgtcagataaagtcccccgtgaactttacccggtggtgcatatcggggatgaaagctggcgcatgatgaccaccgatatggccagtgtgccggtctccgttatcggggaagaagtggctgatctcagccaccgcgaaaatgacatcaaaaacgccattaacctgatgttctggggaatataa

amino acid sequence of ccdB protein: SEQ ID NO.5:

QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKVPRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI

lac UV5promoter sequence:

tttacactttatgcttccggctcgtataatg

(2) Confirmation of TIR key site in Ca.N. koreensis koreensis casposase integration

To determine the TIR sequence involved in Ca.N.koreensis caspase recognition, TIR with different site mutations was incubated with 45bp double-stranded target (consisting of 9bp leader,13bp TSD and 13bp spacer) and Ca.N.koreensis caspase formed by annealing single-stranded DNA labeled with the 3 '-end 6-FAM and 3' -end Cy5, respectively, and key TIR sites involved in the integration reaction were determined by differences in the integration activities of the different mutants. Firstly taking the ssTIR sequence as the WT, and carrying out section-by-section mutation on the sequence, wherein the integration activity of the ssTIR (1-25) mutant is not significantly different from that of the WT; whereas the integration activity at both ends after the 26-30 mutation at the 3' -end of the ssTIR sequence significantly reduced the L-int reduction more significantly; thus, the activity of the 3' -terminal 5' -TTCTA-3' motif in the ssTIR sequence for the integration reaction is most affected, which indicates that the motif is a key region for the recognition of the ssTIR sequence by Ca.N.koreensis caspase, and there is a high probability of important interactions with Ca.N.koreensis caspase.

Although single base mutations of 5'-TTCTA-3' motif do not greatly affect TIR integration, it is speculated that nucleotide sequences located within the motif may have synergistic or interactive effects, together with the efficiency of TIR sequence integration, due to a significant decrease in TIR integration activity following all mutations. The above results indicate that: unlike the a.boost and m.mazei caspases of family type 2, which have stringent requirements for TIR nucleotide sequences involved in integration, ca.n.koreensis caspases have broader compatibility for TIR that can be integrated, which may be closer to Cas1-Cas2 of the CRISPR-Cas system. In addition to ssTIR as described above, the present study also performed similar integration reactions on the dsTIR sequence to confirm that dsTIR possesses the same ca.n.koreensis caspase recognition site as ssTIR during integration of TIR.

Taken together, as shown in FIG. 9, the results of the integration activities of ssTIR and dsTIR mutants indicate that the 5' -TTCTA-3' motif at the 3' -end of the TIR sequence is the most critical region in the integration reaction.

(3) TIR transformation in Ca.N. koreensis caspase integration into ccdB expression reading frame control switch

The 5' end 10nt of the original 20nt TIR of Ca.N. koreensis caspase is modified, so that the Ca.N. koreensis caspase can play different roles in the full integration results in different directions in the Mini-caspase insertion process. The modified TIR is renamed TIRF (25 nt long, upstream TIR) and TIRR (19 nt long, downstream TIR) with the target insertion direction as the reference direction. Compared with the original TIR, the 11 th, 12 th and 19 th bases of the TIRR are mutated, and the mutation of the sites can be changed into ccdB protein ORF in different insertion results to play a role of an initiation codon or a termination codon. The 19 bases near the 3 'end of TIRF are identical to TIRR, and the 6nt near the 5' end is the start codon introduced. As shown in fig. 10, the modified TIRF and TIRR will together with the target gene constitute Mini-cascoson which will close the ORF of ccdB protein after complete integration in the target direction, but will maintain complete expression of ccdB when complete integration in the non-target direction is completed, so that on the screening plate transformed by the tool strain without f+ plasmid, monoclonal colonies corresponding to the products of complete integration in the target direction can normally grow.

The related sequences are shown in the following table:

name of the name	Sequence (5 '-3')	Remarks
			ssTIR30(1-5)	tacgaatgataacggaataaaaagattcta	——
ssTIR30(6-10)	atgcttactaaacggaataaaaagattcta	——
			ssTIR30(11-15)	atgctatgatttgccaataaaaagattcta	——
ssTIR30(16-20)	atgctatgataacggttattaaagattcta	——
			ssTIR30(21-25)	atgctatgataacggaataatttctttcta	——
ssTIR30(1-25)	tacgatactattgccttatttttctttcta	——
			ssTIR30(26-30)	atgctatgataacggaataaaaagaaagat	——
ssTIR30-T26A	atgctatgataacggaataaaaagaatcta	——
			ssTIR30-T27A	atgctatgataacggaataaaaagatacta	——
ssTIR30-C28G	atgctatgataacggaataaaaagattgta	——
			ssTIR30-T29A	atgctatgataacggaataaaaagattcaa	——
ssTIR30-A30T	atgctatgataacggaataaaaagattctt	——
			ssTIR30-A30C	atgctatgataacggaataaaaagattctc	——
ssTIR30-A30G	atgctatgataacggaataaaaagattctg	——
			TIRF	tagaatctttacattcgtaatgcat	SEQ ID NO.6
TIRR	tacgaatgtaaagattcta	SEQ ID NO.7

Example 5

The present example is to test the full integration capacity of the modified type I caspase system in E.coli expression system.

(1) Construction of pltdet-p 3c-Cas2Cas3 vector

Randomly selecting two vectors (pRSF-Duet-Cas 2 and pLenti-Cas3 are selected in the embodiment), amplifying Cas3 protein ORF by a PCR method, carrying out homologous recombination on the protein ORF to the pRSF-Duet-Cas2 vector for recombination, and then transferring the recombinant plasmid into DH5 alpha competence by a chemical transformation method; the correct recombinant plasmid was obtained after Sanger sequencing using plasmid extraction Kit extraction plasmid. Construction of pltdet-p 3c-Cas2Cas3 plasmid structures by the methods described above is shown in fig. 11. The vector is a template vector for preparing Mini-Casboost.

(2) Preparation of Mini-Casboost

The pLtDuet-p3c-Cas2Cas3 vector is used as a template, 1K-7K fragments with different lengths are selected to design upper amplification primers and lower amplification primers, a TIRF sequence with the length of 25nt is added at the 5 'end of the original upstream amplification primer, a TIRR sequence with the length of 19nt is added at the 5' end of the original downstream amplification primer, and pLtDuet-TIRF-F, pLtDuet-TIRR-1K-R, pLtDuet-TIRR-2K-R, pLtDuet-TIRR-3K-R, pLtDuet-TIRR-4K-R, pLtDuet-TIRR-5K-R, pLtDuet-TIRR-6K-R, pLtDuet-TIRR-7K-R primers are obtained, and then PCR amplification is carried out and the PCR products are cut and recovered to obtain the pLtDuet-Mini-caspase with the length of 1K/2K/3K/4K/5K/6K/7K.

The sequence involved is as follows:

pltdet-TIRF sequence:

tagaatctttacattcgtaatgcatcgctgagcaataactagcataacccc

pLtDuet-TIRR-1K-R sequence:

tagaatctttacattcgtaggaagagcgctgcatgcctat

pLtDuet-TIRR-2K-R sequence:

tagaatctttacattcgtactcgaccgatgcccttgagag

pLtDuet-TIRR-3K-R sequence:

tagaatctttacattcgtattaggtgaatgtgaaaccagtaacgttatacg

pLtDuet-TIRR-4K-R sequence:

tagaatctttacattcgtattacggaactcccaagcttatcgataaaattttg

pLtDuet-TIRR-5K-R sequence:

tagaatctttacattcgtattagtctgaggcacgaatcccttgg

pLtDuet-TIRR-6K-R sequence:

tagaatctttacattcgtattatggtccacggtagcgctgaagaa

pLtDuet-TIRR-7K-R sequence:

tagaatctttacattcgtattatccaggtacttaggcagctgagg

1 k-pltdet-Mini-caspaseon sequence:

tagaatctttacattcgtacgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaacctcaggcatttgagaa

tacgaatgtaaagattcta

2 k-pltdet-Mini-caspaseon sequence:

tgtaattctcatgttagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagtacgaatgtaaagattcta

3 k-pltdet-Mini-caspaseon sequence:

tagaatctttacattcgtaatgcatcgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgaaacctcaggcattt

gagaagcacacggtcacactgcttccggtagtcaataaaccggtaaaccagcaatagacataagcggctatttaacgaccctgccctgaaccgacgaca

agctgacgaccgggtctccgcaagtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatga

attaattcttagaaaaactcatcgagcatcaaatgaaactgcaatttattcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggag

aaaactcaccgaggcagttccataggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaa

aaataaggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagtttatgcatttctttccagacttgttcaacaggccagc

cattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagcgagacgaaatacgcggtcgctgttaaaaggacaat

tacaaacaggaatcgaatgcaaccggcgcaggaacactgccagcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgtttt

cccggggatcgcagtggtgagtaaccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctg

accatctcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatagattgtcgcacctg

attgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggcctagagcaagacgtttcccgttgaatatggctc

atactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggcatgcagcg

ctcttccgcttcctcgctcactgactcgctacgctcggtcgttcgactgcggcgagcggtgtcagctcactcaaaagcggtaatacggttatccacagaatc

aggggataaagccggaaagaacatgtgagcaaaaagcaaagcaccggaagaagccaacgccgcaggcgtttttccataggctccgcccccctgacg

agcatcacaaaaatcgacgctcaagccagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctct

cctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgttggtatctcagttcggtgta

ggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaaga

cacgacttatcgccactggcagcagccattggtaactgatttagaggactttgtcttgaagttatgcacctgttaaggctaaactgaaagaacagattttggt

gagtgcggtcctccaacccacttaccttggttcaaagagttggtagctcagcgaaccttgagaaaaccaccgttggtagcggtggtttttctttatttatgaga

tgatgaatcaatcggtctatcaagtcaacgaacagctattccgttactctagatttcagtgcaatttatctcttcaaatgtagcacctgaagtcagccccatacg

atataagttgtaattctcatgttagtcatgccccgcgcccaccggaaggagctgactgggttgaaggctctcaagggcatcggtcgagatcccggtgccta

atgagtgagctaacttacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcgg

ggagaggcggtttgcgtattgggcgccagggtggtttttcttttcaccagtgagacgggcaacagctgattgcccttcaccgcctggccctgagagagttg

cagcaagcggtccacgctggtttgccccagcaggcgaaaatcctgtttgatggtggttaacggcgggatataacatgagctgtcttcggtatcgtcgtatc

ccactaccgagatgtccgcaccaacgcgcagcccggactcggtaatggcgcgcattgcgcccagcgccatctgatcgttggcaaccagcatcgcagt

gggaacgatgccctcattcagcatttgcatggtttgttgaaaaccggacatggcactccagtcgccttcccgttccgctatcggctgaatttgattgcgagtg

agatatttatgccagccagccagacgcagacgcgccgagacagaacttaatgggcccgctaacagcgcgatttgctggtgacccaatgcgaccagatg

ctccacgcccagtcgcgtaccgtcttcatgggagaaaataatactgttgatgggtgtctggtcagagacatcaagaaataacgccggaacattagtgcag

gcagcttccacagcaatggcatcctggtcatccagcggatagttaatgatcagcccactgacgcgttgcgcgagaagattgtgcaccgccgctttacagg

cttcgacgccgcttcgttctaccatcgacaccaccacgctggcacccagttgatcggcgcgagatttaatcgccgcgacaatttgcgacggcgcgtgca

gggccagactggaggtggcaacgccaatcagcaacgactgtttgcccgccagttgttgtgccacgcggttgggaatgtaattcagctccgccatcgccg

cttccactttttcccgcgttttcgcagaaacgtggctggcctggttcaccacgcgggaaacggtctgataagagacaccggcatactctgcgacatcgtat

aacgttactggtttcacattcacctaatacgaatgtaaagattcta

4 k-pltdet-Mini-caspaseon sequence:

aacgttactggtttcacattcaccaccctgaattgactctcttccgggcgctatcatgccataccgcgaaaggttttgcgccattcgatggtgtccgggatctc

gacgctctcccttatgcgactcctgcattaggaaattaatacgactcactataggggaattgtgagcggataacaattcccctgtagaaataattttgtttattt

ggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagattggaatcacacgacctggatgga

gtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattag

ataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagttttt

gctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaa

tagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggttaacttttaaaagaaaaggggggattgggggg

tacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttatcgataagctt

gggagttccgtaatacgaatgtaaagattcta

5 k-pltdet-Mini-caspaseon sequence:

gggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagta

acgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccct

attgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctat

taccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagttt

gttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatata

agcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgactctagtccagtgtggtggaatt

ctgcagatatcaacaagtttgtacaaaaaagcaggctccgcggccgcacagatctcgaggtgtcgtgaacaccgccaccatgggcagcagccatcatc

accaccaccacagccagatgctgaaacagctgctggccaagagcctgcctaccgatcctcagaagaagcccctgagcctggaacagcatctgctgga

cacagagacagccgctctggtcatcttcaagggcagaatgctggacaactggtgccggttcttcaaagtgaaggaccccgacgagttcctgctgcacct

gagagtggccgctctgtttcacgatctgggcaaagccaaccacgagttcatcgaggccgtgaccgccaagggattcgtgcctcagactaatacgaatgt

aaagattcta

6 k-pltdet-Mini-caspaseon sequence:

gagagtggccgctctgtttcacgatctgggcaaagccaaccacgagttcatcgaggccgtgaccgccaagggattcgtgcctcagacactgagacacg

agtggatctctgccctggtgctgcatctgcctgaagttcgacagtggctgggcaagagcaacctgaacctggaagtggttacagccgccgtgctgagcc

accacctgaaagcttctcccgacggcgactacaagtgggacgagcctcagaaaagcggcgacaaggtggaaacaaagctgtacttcaaccacgaaga

ggtggaccggatcctgaacaagatcgccaacctgctggacgtggacagcaagctgcctgagctgcccaagaagtggatcaagggcgacatcttcctg

gaaaacatctacaaggacgccaaccagatcggccggaagttcaccagacaggccaagaaggacgacagcctgaagggactgctgctggctgtgaag

gccggactgatcgcctctgattctgtggccagcggcatctacagaacccaggactctgaggccattgccaactgggtcaaccagacactgcacaccaac

agcatcacccctgaggaaatcgaggaaaagattctgcaccctcggtacagacaggtggaaaagagcatcaacgagcccttccagctgaagcggttcca

agagaaggccgagactctgagcagtcggctgctgctgatgtctggctgtggctctggcaagaccatcttcgcctataagtggatgcagggcgtgctgaa

caagcaccaggccggcagagccatctttctgtaccctacaagaggcaccgccaccgagggcttcaaggactatgtgtcctggtgtcctgaggccgatg

cctctctgctgactggcacagccacatacgagctgcaggctatcgccaagaatcccaccgaggccaacgagggcaaagactaccaggccgacgaga

gactgtacgccctcggctattggggcaagagattcttcagcgctaccgtggaccataatacgaatgtaaagattcta

7 k-pltdet-Mini-caspaseon sequence:

gactgtacgccctcggctattggggcaagagattcttcagcgctaccgtggaccagttcctgagctttctgacccacaactacaagagcatctgtctgctg

cccgtgctggccgatagcgtggtggttatcgatgagatccacagcttcagccccgagatgttcgacagcctcgtgtgcttcctgaaaaccttcgacgtgcc

agtgctgtgcatgaccgctacactgccccagaccagaatcgaggacctgaccatccagctcgacaaggacaaggatggcctgggcctcgaggtgttcc

ctacctctgatagaagcgagctggccgagctggaaaaggccgaaggcatggaaagatacctgatcgcccacaccaacgaggaagctgccctggatct

ggccgtgaaagcctaccaggatagcaagagggtgctgtgggtcgtgaacaccgtggacagatgcagagagaaagcccggaagctggaatgcctgct

gaaaaccgaggtgctgacctaccacagccggttcaaactggccgaccggcagaacagacaccgggaaaccgtggaagccttcgctctgcatcaggc

ccagggcgagaaaaaagccgccattgccgtgaccacacaagtgtgcgagatgtccctggacctggacgccgatgtgctgatcacagagctggcccct

atcagcagcctggtgcagagattcggcagaagcaaccggggcgacaagaacgacaagaccgagcctagcaagatctacgtgtacaagcctcctaag

gacaagccctacaagcagaaggatgatctggaccccgccgagaagttcatcaacgacgttctgggcagagcctctcagaagctcctggccgagaagc

tgaaagagcacagccctccaggcagatactccgatggatctgcccctttcgtgacccaaggctactgggcctctagcgacgagcctttcagaaagatcg

acgacttcgccgtgaacgcagtgctgacagaggatctgggcgagatcacccagtacctgaacagcaaccctcctaagcctatcgacggcttcatcgtgc

ccgtgcctaagaagtacaagttccagggcttcagccaccggccacctcagctgcctaagtacctggataatacgaatgtaaagattcta

(3) Verifying the full integration ability of the modified Ca.N.koreensis strain type I caspase gene insertion tool

The resulting pltdet-Mini-caspases were incubated with Ca.N. koreensis caspase and pET28a-lac UV5 proter-Ca.N. koreensis target-ccdB vector to catalyze the integration reaction of pltdet-Mini-caspases. After isopropanol precipitation, the reacted product was electrotransferred to an ElectroMax E.coli DH10B competence and plated onto kanamycin sulfate-containing resistant plates. Transformants grown on the resistant plates were the product of the full integration reaction, and several monoclonal were randomly picked for Sanger sequencing.

The specific process is as follows: each pLtDuet-Mini-caspases (final concentration 0.2. Mu.M), ca.N.koreensis caspase (final concentration 1. Mu.M), pET28a-lac UV5 proter-Ca.N.koreensis target-ccdB (final concentration 65 ng/. Mu.L) was measured at 1X Integration buffer (20mM HEPEs pH 7.5,150mM NaCl,5mM MnCl) ₂ And 50. Mu.g/mL BSA) at 37℃for 60min to catalyze the integration of pLtDuet-Mini-casosons, followed by the addition of 50mM EDTA to terminate the reaction. The integrated product was recovered by the PCR product recovery kit and then dissolved in about 8. Mu.L of sterile water. Adding 5 mu L of recovered product into 50 mu L of Thermo DH10B commercial electric transfer competence, carrying out ice bath for 2min, transferring to an electric transfer cup with a hole width of 1mm for ice bath in advance, carrying out electric shock on a Bio-Rad electric transfer instrument (GenePulser) according to 2.5kV/cm, electric resistance of 200 omega and capacitance of 25 mu F for 5ms, carrying out ice bath for 30s, adding 800 mu L of preheated SOC non-resistant culture medium, culturing at 37 ℃ for 1h at 200rpm, coating bacterial liquid on a resistance plate containing kanamycin, culturing at 37 ℃ for 20h, and randomly picking a plurality of monoclonal to carry out Sanger sequencing, wherein the monoclonal which can normally grow is the product of full-integration reaction. ( And (3) injection: the corresponding steps of example 3 and example 4 employ the same fully integrated process parameters or conditions as in this example. )

The results are shown in FIG. 12: the expected pltdetet-Mini-caspases exist in the plasmid after the integration reaction, the integration of 7 k-pltdetet-Mini-caspases can be realized at the longest and the insertion is in the target direction, and the result proves that the I-type caspase gene insertion tool of the modified Ca.N.koreensis strain can realize the fixed target point fixed direction insertion and has the advantage of carrying long fragment integration.

By combining the above embodiments, the invention mainly provides a type I caspase gene insertion tool derived from Ca.N.koreensis strain and application thereof, which not only can realize the fixation direction insertion of a target gene fixation target, but also can ensure that double-stranded DNA is not broken in the editing process to reduce the hidden trouble existing in off-target and other single-stranded gene editing technologies, and has the integration capacity of 7k, which shows the superiority of the system and has great research and development potential.

In addition to the embodiments described above, other embodiments of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.

Claims

1. A type I caspase gene insertion tool is characterized by comprising a caspase protein, mini-caspaseon containing target gene fragments and target-ccdB vector; the amino acid sequence of the caspase protein is SEQ ID NO.2; the Mini-Caspo is dsDNA, and has the structure that one end of a target gene fragment is connected with a TIRF sequence, the other end of the target gene fragment is connected with a TIRR sequence, the nucleotide sequence of the TIRF sequence is SEQ ID NO.6, and the nucleotide sequence of the TIRR sequence is SEQ ID NO.7; the target-ccdB vector contains a target gene fragment and a coding gene fragment of ccdB protein, the sequence of the target gene fragment is SEQ ID NO.3, the amino acid sequence of the ccdB protein is SEQ ID NO.5, and the coding gene fragment of the ccdB protein corresponds to the amino acid sequence of the ccdB protein.

2. The tool for inserting a type I caspase gene according to claim 1, wherein the Mini-caspase comprises a TIRF sequence, a target gene fragment and a TIRR sequence from upstream to downstream; the target-ccdB vector contains the lac UV5 promoter.

3. The tool for inserting a type I caspase gene according to claim 1, wherein the gene sequence encoding the caspase protein is SEQ ID No.1; the coding gene fragment sequence of the ccdB protein is SEQ ID NO.4.

4. The tool for inserting a type I caspase according to claim 1, wherein said caspase protein and target gene fragment is derived from ca.n.koreens, respectively.

5. A method of gene insertion, characterized in that a type I caspase gene insertion tool according to any one of claims 1-4 is used;

the gene insertion method comprises the following steps:

6. The method for gene insertion according to claim 5, wherein in the first step, the caspase protein is prepared by the steps of:

s2, performing induction culture on strains containing the plasmids, and purifying by crushing thalli, centrifuging and nickel column affinity chromatography to obtain caspase protein.

7. The method for gene insertion according to claim 5, wherein the step of preparing Mini-casnoson containing the target gene fragment comprises the steps of:

designing an upstream amplification primer and a downstream amplification primer according to a target gene fragment, adding a TIRF sequence at the 5 'end of the upstream amplification primer, adding a TIRR sequence at the 5' end of the downstream amplification primer to obtain a specific primer, amplifying Mini-Caspo with a structure of the TIRF sequence-target gene fragment-TIRR sequence by using the specific primer through a PCR (polymerase chain reaction) mode, and finally obtaining purified Mini-Caspo by a gel cutting recovery mode.

8. The method of gene insertion according to claim 6, wherein in the first step, the target-ccdB vector is pET28a-lac UV5promoter-Ca.N.koreensis target-ccdB; in S1, the plasmid containing the coding gene sequence of the caspase protein is pET-28a-ts-sumo-Ca.N. koreensis-caspase.

9. The method according to claim 5, wherein in the second step, the final concentration of caspase protein is 1.+ -. 0.1. Mu.M, the final concentration of Mini-Caspo is 0.2.+ -. 0.05. Mu.M, and the final concentration of target-ccdB vector is 65.+ -. 5 ng/. Mu.L; the reaction conditions are that the reaction is carried out for at least 60min at 37+/-0.5 ℃ under the condition of 1X Integration buffer; EDTA was added at the termination of the reaction to terminate the reaction; recovering the integrated product by using a PCR product recovery kit;

in the third step, the competent cell is DH10B; when the electric power is turned, an electric power turning instrument is adopted, and electric shock parameters are as follows: 2.5kV/cm, resistance 200 omega, capacitance 25 muF, time 5ms; after electrotransformation, culturing in SOC non-antibiotic liquid culture medium, and then coating bacterial liquid in kanamycin sulfate-containing resistance plate for culturing, wherein the normal monoclonal is the gene integration product containing target gene fragment.

10. Use of a type I caspase gene insert tool according to any one of claims 1-4 for editing e.coli genes.