Multi-site chromosome editing tool and use thereof

Abstract

Provided are a multi-site chromosome editing tool and the use thereof, relating to tools such as Nox variants, DNA molecule sets, and circular vectors. Specifically, a Nigri recombinase is used to recognize and mediate the recombination of Nox sites (containing Nox and variants Nox 4-5 and 8-11 thereof), and a Cre recombinase is used to mediate the rearrangement of Lox sites. By means of specific recombination of knock-in sites and adjacent knock-out sites, the targeted, continuous, and multi-site integration of large fragments of circular DNA at specific sites in the genome is achieved, and CRISPR technology can also be combined to assist integration.

Description

A multi-site chromosome editing tool and its applications

[0001] Cross-referencing of related applications:

[0002] This application claims priority to Chinese patent applications filed on April 16, 2025 (application number 202510477063.4), June 10, 2025 (application number 202510771253.7), and January 23, 2026 (application number 202610092929.4), the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of biotechnology, specifically to a multi-site chromosome editing tool and its applications. Background Technology

[0004] In the field of biotechnology research, constructing highly efficient engineered strains (cells) is a core step in realizing the industrial production of biofuels, biomaterials, and high-value-added chemicals. Optimizing strain traits is a multi-dimensional systems engineering process requiring precise manipulation of the genome. To achieve a step-by-step improvement in strain performance, researchers often need to perform multiple rounds of differentiated gene integration and knockout operations at multiple sites on the genome. This process not only requires scientific evaluation of existing genetic modification effects but also the development of continuous genome editing platforms with iterative optimization capabilities.

[0005] An ideal genome manipulation system should possess the following key characteristics: delivery capability of ultra-large capacity heterologous DNA fragments (>10kb), flexibility in multi-target collaborative editing, near-saturation integration efficiency, modular and rapid reprogramming characteristics, and highly standardized molecular operation procedures, thereby effectively overcoming the limitations of traditional technologies in multi-round iterative optimization. Simultaneously, an ideal editing tool should also be able to achieve stable, precise, and continuously operable large-fragment DNA integration in various eukaryotic systems, meeting the application needs of multiple scenarios such as plant trait improvement, stable animal cell line construction, and eukaryotic microbial pathway optimization.

[0006] Currently, commonly used gene editing technologies mainly fall into three categories: zinc finger nuclease-mediated gene editing (ZFN), transcription activator-like effector nuclease-mediated gene editing (TALEN), and clustered short palindromic repeat-mediated gene editing (CRISPR-Cas system). The core mechanism of these technologies relies on sequence-specific nucleases introducing DNA double-strand breaks (DSBs) at the target site, thereby achieving gene knockout or integration of exogenous sequences through homologous directed repair (HDR) or non-homologous end joining (NHEJ) pathways in the host cell.

[0007] Technological innovations, exemplified by the CRISPR system, have spawned various advanced strategies. For instance, combining CRISPR with lead editing enables precise base substitutions or fragment insertions at specific sites in the eukaryotic genome. Meanwhile, CRISPR-guided transposase systems (such as INTEGRATE) have pioneered a new paradigm for RNA-guided site-specific integration of DNA fragments. Despite significant advancements in current technologies, many fundamental challenges remain in genome editing in both prokaryotes and eukaryotes.

[0008] In prokaryotes, homologous recombination efficiency is extremely low in most prokaryotes, and commonly used gene editing methods have significant shortcomings. Although λ-red and RecET-mediated recombination systems are widely used and can be combined with the CRISPR-Cas9 system to improve recombination efficiency, their recombination efficiency is strictly dependent on the length of the homologous arm and the length of the exogenous integration fragment. When integrating multiple gene clusters, multiple rounds of integration processes are required. The CRISPR RNA-guided transposases (CAST) method can achieve site-specific integration of large fragments (>10kb) and simultaneous editing of multiple sites, but it leaves the same LR / ER sequences at different sites in the genome, which can easily reduce genome stability and lead to large-fragment DNA deletion.

[0009] While CRISPR systems exhibit high editing efficiency in eukaryotes, they still have limitations in integrating large DNA fragments and performing continuous multi-round editing, with efficiency significantly decreasing when integrating gene clusters exceeding several kb in length. More critically, HDR efficiency is low in most eukaryotic systems, a problem particularly pronounced in non-dividing cells or difficult-to-transfect cell types (such as neurons, stem cells, and some protists). Although introducing exogenous recombinases or optimizing delivery systems can improve integration efficiency to some extent, it is still insufficient to meet the large-capacity, multi-site integration requirements for constructing complex metabolic pathways or integrating multi-gene circuits. Furthermore, the λ-Red and RecET homologous recombination systems widely used in prokaryotes cannot function effectively in eukaryotic systems. In addition, existing editing systems generally rely on the synergistic effects of multi-component protein complexes, which not only reduces editing efficiency but also increases the metabolic burden on host cells, ultimately affecting the physiological performance of engineered strains.

[0010] In recent years, site-specific recombinase (SSR)-mediated recombination systems have emerged as a potential breakthrough in overcoming the aforementioned bottlenecks. This system represents the third type of recombination process in bacteria, in addition to homologous recombination and transposition. It catalyzes recombination reactions between two short conserved sequences in different DNA molecules, enabling the integration or excision of exogenous DNA elements. Site-specific recombinases are mainly divided into two categories: tyrosine and serine. Tyrosine recombinases, represented by Cre and Flp, can recognize short, specific sites (loxP, FRT) of 34 bp, and the reaction does not require cofactors. They can catalyze sequence-specific DNA recognition, cleavage, and rejoining reactions through a single protein, and have been widely used in gene regulation and element integration in eukaryotic systems. However, these recombinases lack the ability to control the integration direction, making it difficult to achieve efficient integration of large, continuous fragments, thus limiting their application in complex genome reconstruction. Serine recombinases can mediate recombination between attP and attB sites, forming attL and attR sites. However, their recognition sites are relatively long, and the integrase protein has a large molecular weight, leading to difficulties in cell delivery. Furthermore, the attL and attR sites formed by recombination cannot be repeatedly recognized by the same integrase, making continuous gene editing difficult.

[0011] In summary, existing genome editing technologies have significant shortcomings in terms of large fragment carrying capacity, multi-target synergy, and continuous iterative operation. Developing a new generation genome editing platform that is applicable to both prokaryotic and eukaryotic systems, has ultra-large DNA loading capacity, high integration efficiency, and modular rapid reprogramming characteristics has become an urgent need in fields such as synthetic biology, metabolic engineering, and gene therapy.

[0012] Invention Overview

[0013] This invention claims protection for a kit (composition) for integrating or continuously integrating the genome of a host cell.

[0014] The complete product (composition) is either a complete product 1 (composition 1) for integrating the genome of a host cell or a complete product 2 (composition 2) for continuously integrating the genome of a host cell.

[0015] The complete product 1 (composition 1) comprises (or is composed of): (A1), (A3) and (A4); or, the complete product 1 (composition 1) comprises (or is composed of): (A2), (A3) and (A4).

[0016] The complete product 2 (composition 2) includes (or is composed of): (A1), (A2), (A3) and (A4).

[0017] (A1) Circular vector 1; the circular vector 1 is composed of the following elements in the order from 5' to 3': loxF1, nox, replication origin site, resistance gene, lox71, P3, P1, P2;

[0018] (A2) Circular vector 2; the circular vector 2 is composed of the following elements in the order from 5' to 3': lox66, nox, replication origin site, resistance gene, loxF2, P3, P1, P2;

[0019] (A3) Circular vector 3; the circular vector 3 is used to express Nigri recombinase and Cre recombinase;

[0020] (A4) A site-specific knock-in tool; the site-specific knock-in tool is capable of knocking in a specific DNA fragment at the integration site of the host cell’s genome; the specific DNA fragment consists of lox66 and nox or loxF1 and nox (the specific DNA fragment contains the following elements in 5' to 3' order: lox66 and nox, or loxF1 and nox).

[0021] The integration can be either insertion or replacement.

[0022] The loxF1 and loxF2 are recognition sites of Cre recombinase. Under the action of Cre recombinase, they can recombine to achieve the purpose of deleting the fragment between the two sites (the site formed after recombination is inactive, that is, it cannot be recognized by Cre recombinase again to recombine). The lox66 and lox71 are also recognition sites of Cre recombinase. Under the action of Cre recombinase, they can recombine to achieve the purpose of deleting the fragment between the two sites (the site formed after recombination is inactive, that is, it cannot be recognized by Cre recombinase again to recombine). Furthermore, the two pairs of recognition sites, loxF1 / loxF2 and lox66 / lox71, are independent of each other and will not undergo cross-recombination.

[0023] P1 and P2 are adapter sequences, and the position between P1 and P2 is used for inserting the DNA fragment to be integrated; P3 is a universal primer sequence used for PCR detection of the recombinant vector obtained after inserting the DNA fragment to be integrated.

[0024] The host cell may be bacteria, fungi, animal cells, or plant cells, etc.

[0025] Furthermore, the host cell may be Escherichia coli.

[0026] In one embodiment of the present invention, the replication initiation site is oriR6k in both the annular carrier 1 and the annular carrier 2.

[0027] Furthermore, in the circular vector 1 and the circular vector 2, the resistance gene is preferably different, but it can also be the same.

[0028] The resistance gene can be selected from: kanamycin resistance gene, streptomycin resistance gene, etc.

[0029] Further, the nucleotide sequence of loxF1 is SEQ ID NO:1; the nucleotide sequence of nox is SEQ ID NO:2; the nucleotide sequence of oriR6k is SEQ ID NO:3; the nucleotide sequence of lox71 is SEQ ID NO:5; the nucleotide sequence of P3 is SEQ ID NO:6; the nucleotide sequence of P1 is SEQ ID NO:7; the nucleotide sequence of P2 is SEQ ID NO:8; the nucleotide sequence of lox66 is SEQ ID NO:9; and the nucleotide sequence of loxF2 is SEQ ID NO:10.

[0030] The amino acid sequence of the Nigri recombinase is identical to the amino acid sequence obtained by translating SEQ ID NO:15. The amino acid sequence of the Cre recombinase is identical to the amino acid sequence obtained by translating SEQ ID NO:13.

[0031] In one embodiment of the present invention, the circular vector 1 is composed of the following elements in the order from 5' to 3': loxF1, nox, oriR6k, Kan (kanamycin resistance gene), lox71, P3, P1, and P2. The nucleotide sequence of the Kan (kanamycin resistance gene) is SEQ ID NO:4. Specifically, the circular vector 1 is pKNF-1; the complete sequence of pKNF-1 from the 5' end to the 3' end is "SEQ ID NO:1+SEQ ID NO:2+SEQ ID NO:3+SEQ ID NO:4+SEQ ID NO:5+SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8".

[0032] In another embodiment of the present invention, the circular vector 1 is composed of the following elements in the order from 5' to 3': loxF1, nox, oriR6k, Str (streptomycin resistance gene), lox71, P3, P1, and P2. The nucleotide sequence of the Str (streptomycin resistance gene) is SEQ ID NO:22. Specifically, the circular vector 1 is pSNF-1; the complete sequence of pSNF-1 from the 5' end to the 3' end is "SEQ ID NO:1+SEQ ID NO:2+SEQ ID NO:3+SEQ ID NO:22+SEQ ID NO:5+SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8".

[0033] In one embodiment of the present invention, the circular vector 2 is composed of the following elements in the order from 5' to 3': lox66, nox, oriR6k, Str (streptomycin resistance gene), loxF2, P3, P1, and P2; wherein the nucleotide sequence of Str (streptomycin resistance gene) is SEQ ID NO:22. Specifically, the circular vector 2 is pSNL-1; the full sequence of pSNL-1 from the 5' end to the 3' end is "SEQ ID NO:9+SEQ ID NO:2+SEQ ID NO:3+SEQ ID NO:22+SEQ ID NO:10+SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8".

[0034] In another embodiment of the present invention, the circular vector 2 is composed of the following elements in the order from 5' to 3': lox66, nox, oriR6k, Kan (kanamycin resistance gene), loxF2, P3, P1, and P2; wherein the nucleotide sequence of the Kan (kanamycin resistance gene) is SEQ ID NO:4. Specifically, the circular vector 2 is pKNL-1; the full sequence of pKNL-1 from the 5' end to the 3' end is "SEQ ID NO:9+SEQ ID NO:2+SEQ ID NO:3+SEQ ID NO:4+SEQ ID NO:10+SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8".

[0035] Furthermore, the circular vector 3 contains an expression cassette 1 for expressing the Cre recombinase, an expression cassette 2 for expressing the Nigri recombinase, and a replication initiation site.

[0036] Furthermore, in expression cassette 1, the promoter for initiating transcription of the gene encoding the Cre recombinase is ParaBAD, and the termination sequence is TrrnB; in expression cassette 2, the promoter for initiating transcription of the gene encoding the Nigri recombinase is Ptet, and the termination sequence is TrrnB; the replication initiation site is RK2s, which is a temperature-sensitive RK2 replication initiation site.

[0037] More specifically, the circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, the resistance gene, and RK2s.

[0038] The resistance gene in the circular vector 3 is different from the resistance genes in the circular vector 1 and the circular vector 2.

[0039] The nucleotide sequence of ParaBAD is SEQ ID NO:16; the nucleotide sequence of the gene encoding Cre recombinase is SEQ ID NO:13; the nucleotide sequence of TrrnB is SEQ ID NO:20; the nucleotide sequence of Ptet is SEQ ID NO:19; the nucleotide sequence of the gene encoding Nigri recombinase is SEQ ID NO:15; and the nucleotide sequence of RK2s is SEQ ID NO:21.

[0040] In one embodiment of the present invention, the circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, Amp (ampicillin resistance gene), and RK2s. The nucleotide sequence of Amp (ampicillin resistance gene) is SEQ ID NO:23. Specifically, the circular vector 3 is pW-NC01; the full sequence of pW-NC01 from the 5' end to the 3' end is "SEQ ID NO:16+SEQ ID NO:13+SEQ ID NO:20+SEQ ID NO:19+SEQ ID NO:15+SEQ ID NO:20+SEQ ID NO:23+SEQ ID NO:21".

[0041] Further, the targeted knock-in tool comprises the following: 1) a targeting vector containing a coding sequence of sgRNA targeting the integration site in the host cell's genome; 2) a homologous recombination fragment containing an upstream homologous arm, the specific DNA fragment, and a downstream homologous arm from the 5' to the 3' end; the upstream homologous arm is a fragment (approximately 1000 bp) located upstream of the integration site in the host cell's genome; the downstream homologous arm is a fragment (approximately 1000 bp) located downstream of the integration site in the host cell's genome; and 3) an expression vector for expressing the Cas protein and the λ-red element.

[0042] In one embodiment of the present invention, the host cell is *Escherichia coli*, and the integration site is located at the position of the *lpxM* gene in *E. coli*, replacing the *lpxM* gene (lipid A biosynthesis myristyltransferase gene). In the site-directed knock-in tool, the targeting vector in 1) is pTarget-lpxM; pTarget-lpxM is a recombinant plasmid obtained by replacing positions 27-246 of plasmid pTargetF with the coding sequence of the sgRNA targeting the *lpxM* gene shown in SEQ ID NO:25. The sequence of the homologous recombination fragment in 2) is SEQ ID NO:26. The expression vector used to express the Cas protein and λ-red element in 3) is the pCAS plasmid. In other embodiments of the present invention, the host cell is Escherichia coli, and the integration site is the lpxL gene, poxB gene, pflB gene, or ldhA gene in Escherichia coli, which is to replace the corresponding gene (corresponding to the process of constructing strains BN-02, BN-03, BN-04, and BN-05 in Example 1).

[0043] Furthermore, the targeted knock-in tool is composed of 1) and 2) mentioned above; and the ring carrier 3 also contains an expression cassette 3 for expressing the Cas protein and an expression cassette 4 for expressing the λ-red element (corresponding to Example 7).

[0044] Specifically, in expression cassette 3, the promoter for initiating transcription of the gene encoding the Cas protein is Psal, and the termination sequence is TrrnB; and / or, in expression cassette 4, the promoter for initiating transcription of the gene encoding the λ-red element (exo-bet-gam gene) is Psal.

[0045] Furthermore, the circular vector 3 is composed of the following elements in the order from 5' to 3': Psal, the gene encoding the Cas protein, TrrnB, Psal, the exo-bet-gam gene, the TλtL3 coding sequence, the ColRNA coding sequence, ParaBAD, the gene encoding the Cre recombinase, TrrnB, Ptet, the gene encoding the Nigri recombinase, TrrnB, the resistance gene, and the RK2s.

[0046] The resistance gene in the circular vector 3 is different from the resistance genes in the circular vector 1 and the circular vector 2.

[0047] The nucleotide sequence of Psal is SEQ ID NO:17; the encoding gene of the Cas protein is GenBank:MW718993.1, positions 3127-7233 (+) (2021-7-20); the nucleotide sequence of TrrnB is SEQ ID NO:20; the exo-bet-gam gene is GenBank:KY563785.1, positions 7320-9224 (+) (2020-4-6); the encoding sequence of TλtL3 is GenBank:MF287367.1, positions 3129-3373 (+) (2017-7-18); the encoding sequence of ColRNA is GenBank:LC377574.3, positions 6930-8489 (-) (2019-8-27); and the nucleotide sequence of ParaBAD is SEQ ID NO:17. NO:16; the nucleotide sequence of the gene encoding the Cre recombinase is SEQ ID NO:13; the nucleotide sequence of the Ptet is SEQ ID NO:19; the nucleotide sequence of the gene encoding the Nigri recombinase is SEQ ID NO:15; the nucleotide sequence of the RK2s is SEQ ID NO:21.

[0048] In one embodiment of the present invention (Example 7), the circular vector 3 is composed of the following elements in the order from 5' to 3': Psal, the gene encoding the Cas protein, TrrnB, Psal, the exo-bet-gam gene, the TλtL3 coding sequence, the ColRNA coding sequence, ParaBAD, the gene encoding the Cre recombinase, TrrnB, Ptet, the gene encoding the Nigri recombinase, TrrnB, Amp (ampicillin resistance gene), and RK2s. The nucleotide sequence of the Amp (ampicillin resistance gene) is SEQ ID NO:23. Specifically, the annular carrier 3 is pW-NC02; the complete sequence of pW-NC02 from the 5' end to the 3' end is “SEQ ID NO:17+GenBank:MW718993.1(3127-7233(+), 2021-7-20)+SEQ ID NO:20+SEQ ID NO:17+GenBank:KY563785.1(7320-9224(+), 2020-4-6)+GenBank:MF287367.1(3129-3373(+)2017-7-18)+GenBank:LC377574.3(6930-8489(-)2019-8-27)+SEQ ID NO:16+SEQ ID NO:13+SEQ ID NO:20+SEQ ID NO:19+SEQ ID NO:15+SEQ ID NO:20+SEQ ID NO:23+SEQ ID NO:21”.

[0049] This invention claims protection for a method for integrating or sequentially integrating the genome of a host cell.

[0050] The method for integrating the genome of a host cell claimed in this invention is achieved using the aforementioned product 1 (composition 1); the method may include the following steps:

[0051] (a1) The specific DNA fragment is knocked into the integration site in the genome of the host cell using the site-specific knock-in tool (based on CRISPR-Cas9 technology).

[0052] (a2) Connect the adapter sequences to both ends of the DNA fragment to be integrated, i.e., connect P1 to one end and P2 to the other end; clone the DNA fragment to be integrated into the circular vector 1 or the circular vector 2 between P1 and P2 using homologous recombination to obtain a recombinant vector (corresponding to pKNF-MVA1 in Example 3); during the construction of the recombinant vector, PCR amplification is performed using P3 as a universal primer and another specific primer for the DNA fragment to be integrated to identify whether the recombinant vector has been successfully constructed.

[0053] (a3) The circular carrier 3 is introduced into the host cell after (a1) treatment.

[0054] (a4) The recombinant vector obtained in (a2) is introduced into the host cell treated in (a3), and after culturing and screening, recombinant cells with one copy of the DNA fragment to be integrated knocked into the integration site are obtained.

[0055] Principle: Nigri recombinase recognizes a nox-specific site and integrates the recombinant vector carrying the nox site into the pre-knocked-in nox site on the host cell genome. Then, Cre recombinase recognizes the pre-knocked-in lox66 site on the host genome and the lox71 site from the circular vector 1 (in which case the specific DNA fragment carried on the site-directed knock-in tool consists of lox66 and nox), or recognizes the pre-knocked-in loxF1 site on the host genome and the loxF2 site from the circular vector 2 (in which case the specific DNA fragment carried on the site-directed knock-in tool consists of loxF1 and nox), and mediates two-site recombination, thereby deleting the replication origin site, resistance gene, and a nox site located between the two sites.

[0056] The method for continuous integration of the host cell genome claimed in this invention is achieved using the aforementioned product 2 (composition 2); the method may include the following steps:

[0057] (b1) The specific DNA fragment is knocked into the integration site in the genome of the host cell using the site-specific knock-in tool (based on CRISPR-Cas9 technology).

[0058] (b2) Connector sequences are attached to both ends of all the DNA fragments to be integrated, i.e., one end is connected to P1 and the other end is connected to P2; after numbering the DNA fragments to be integrated, homologous recombination is used to clone the DNA fragments to be integrated with odd numbers into the circular vector 1 between P1 and P2 to obtain several recombinant vectors 1 (corresponding to pKNF-MVA1 in Example 3), each recombinant vector 1 carrying one DNA fragment to be integrated; homologous recombination is used to clone the DNA fragments to be integrated with even numbers into the circular vector 2 between P1 and P2 to obtain several recombinant vectors 2 (corresponding to pSNL-MVA2 in Example 3), each recombinant vector 2 carrying one DNA fragment to be integrated; during the construction of the recombinant vectors 1 and 2, PCR amplification is performed using P3 as a universal primer and another specific primer for the DNA fragment to be integrated, which can identify whether the recombinant vectors 1 and 2 have been successfully constructed.

[0059] (b3) The circular carrier 3 is introduced into the host cell after treatment (b1).

[0060] (b4) Introduce either recombinant vector 1 or recombinant vector 2 obtained in (b2) into the host cell treated in (b3), and after culturing and screening, obtain a recombinant cell with the first copy of the DNA fragment to be integrated knocked into the integration site (principle: Nigri recombinase recognizes the nox-specific site and integrates the recombinant vector 1 or recombinant vector 2 carrying the nox site into the host cell genome at the pre-knocked nox site; then, Cre recombinase recognizes the lox66 site pre-knocked into the host genome and the lox71 site from the recombinant vector 1, or recognizes the loxF1 site pre-knocked into the host genome and the loxF2 site from the recombinant vector 2, and mediates two-site recombination, thereby deleting the replication initiation site, resistance gene, and one nox site located between the two sites), denoted as recombinant cell 1; Introduce the other recombinant vector 2 or recombinant vector 1 obtained in (b2) into the recombinant cell 2, and after culturing and screening, obtain a recombinant cell with the first copy of the DNA fragment to be integrated knocked into the integration site at ... A recombinant cell with one copy of the first DNA fragment to be integrated and one copy of the second DNA fragment to be integrated simultaneously knocked into the integration site (principle: Nigri recombinase recognizes a nox-specific site and integrates the recombinant vector 2 or the recombinant vector 1 carrying the nox site into the remaining nox site on the genome of the recombinant cell 1; then, Cre recombinase recognizes the loxF1 site retained on the recombinant cell 1 from the recombinant vector 1 and the loxF2 site from the recombinant vector 2, or recognizes the lox66 site retained on the recombinant cell 1 from the recombinant vector 2 and the lox71 site from the recombinant vector 1, and mediates two-site recombination, thereby deleting the replication initiation site, the resistance gene, and a nox site located between the two sites), is designated as recombinant cell 2; in this step, if the first insertion is the recombinant vector 1, then the second insertion is the recombinant vector 2; if the first insertion is the recombinant vector 2, then the second insertion is the recombinant vector 1.

[0061] (b5) Referring to the method in (b4), in the alternating order of introducing the recombinant vector 1 and the recombinant vector 2, all the remaining recombinant vector 1 and all the remaining recombinant vector 2 are sequentially introduced into the recombinant cells obtained in the previous step. The final recombinant cells are the recombinant cells in which one copy of all the DNA fragments to be integrated is knocked into the integration site at the same time.

[0062] The host cell may be bacteria, fungi, animal cells, or plant cells, etc.

[0063] Furthermore, the host cell may be Escherichia coli.

[0064] The method may also include the step of eliminating the circular vector 3 (using the temperature-sensitive replication initiation site carried thereon) in the final recombinant cells by changing the culture temperature.

[0065] In one embodiment of the present invention, in step (a4) or (b4), the culture and screening are performed according to a method including the following steps: the host cells introduced into the recombinant vector 1 or the recombinant vector 2 are grown and revived for 2 hours, then the Cre recombinase expression is induced for 2 hours, and plated; single clones are picked, grown, identified, and preserved (corresponding to scheme 2 in Example 4).

[0066] This invention claims protection for a kit for constructing targeting plasmids required for large-fragment or sequential integration of the host cell genome.

[0067] The kit is kit 1, used to construct a target plasmid for large-fragment integration of the host cell genome, or kit 2, used to construct a target plasmid for continuous large-fragment integration of the host cell genome.

[0068] The kit 1 comprises (or consists of) (B1) and (B3); or, the kit 1 comprises (or consists of) (B2) and (B3).

[0069] The kit 2 comprises (or consists of) (B1), (B2) and (B3).

[0070] (B1) Circular vector 1; the circular vector B1 is composed of the following elements in the order of 5' to 3': loxF1, replication origin site ColE, resistance gene 1, loxF, nox, replication origin site oriSC101, resistance gene 2, lox71, P3, P1, P2.

[0071] (B2) Circular vector 2; the circular vector B2 is composed of the following elements in the order of 5' to 3': lox66, replication origin site ColE, resistance gene 1, loxP, nox, replication origin site oriSC101, resistance gene 2, loxF2, P3, P1, P2.

[0072] (B3) Knock-in tool 1; the knock-in tool 1 is capable of knocking in (inserting or replacing) a specific DNA fragment 1 at a certain site in the genome of the host cell; the specific DNA fragment 1 contains an expression cassette 1 for expressing Cre recombinase and an expression cassette 2 for expressing repA1 protein.

[0073] P1 and P2 are adapter sequences, and the position between P1 and P2 is used to insert the DNA fragment to be integrated. P3 is a universal primer sequence.

[0074] Furthermore, the complete product (composition) also contains the host cell.

[0075] The host cell may be bacteria, fungi, animal cells, or plant cells, etc.

[0076] Furthermore, the host cell may be Escherichia coli.

[0077] The loxF1 and loxF are recognition sites of Cre recombinase. Under the action of Cre recombinase, they can recombine to achieve the purpose of deleting the fragment between the two sites (leaving a loxF1 site after recombination). The lox66 and loxP are also recognition sites of Cre recombinase. Under the action of Cre recombinase, they can recombine to achieve the purpose of deleting the fragment between the two sites (leaving a lox66 site after recombination). Furthermore, the two pairs of recognition sites, loxF1 / loxF and lox66 / loxP, are independent of each other and will not undergo cross-recombination.

[0078] P1 and P2 are adapter sequences, and the position between P1 and P2 is used for inserting the DNA fragment to be integrated; P3 is a universal primer sequence used for PCR detection of the recombinant vector obtained after inserting the DNA fragment to be integrated.

[0079] Furthermore, in the circular vector 1 and the circular vector 2, the resistance gene 1 and the resistance gene 2 are different. The resistance gene 1 and the resistance gene 2 can be selected from: kanamycin resistance gene, streptomycin resistance gene, tetracycline resistance gene, etc.

[0080] Further, the nucleotide sequence of loxF1 is SEQ ID NO:1; the nucleotide sequence of the replication origin site ColE is SEQ ID NO:31; the nucleotide sequence of loxF is SEQ ID NO:34; the nucleotide sequence of nox is SEQ ID NO:2; the nucleotide sequence of the replication origin site oriSC101 is SEQ ID NO:33; the nucleotide sequence of lox71 is SEQ ID NO:5; the nucleotide sequence of P3 is SEQ ID NO:6; the nucleotide sequence of P1 is SEQ ID NO:7; the nucleotide sequence of P2 is SEQ ID NO:8; the nucleotide sequence of lox66 is SEQ ID NO:9; the nucleotide sequence of loxP is SEQ ID NO:35; and the nucleotide sequence of loxF2 is SEQ ID NO:10.

[0081] The amino acid sequence of the Cre recombinase is identical to the amino acid sequence obtained by translating SEQ ID NO:13. The amino acid sequence of the repA1 protein is identical to the amino acid sequence obtained by translating SEQ ID NO:30.

[0082] In one embodiment of the present invention, the circular vector 1 is composed of the following elements in the order from 5' to 3': loxF1, the replication origin site ColE, tetA (tetracycline resistance gene), loxF, nox, the replication origin site oriSC101, Kan (kanamycin resistance gene), lox71, P3, P1, and P2. The nucleotide sequence of tetA (tetracycline resistance gene) is SEQ ID NO:32, and the nucleotide sequence of Kan (kanamycin resistance gene) is SEQ ID NO:4. Specifically, the circular carrier 1 is pKNF-2; the complete sequence of pKNF-2 from the 5' end to the 3' end is "SEQ ID NO:1+SEQ ID NO:31+SEQ ID NO:32+SEQ ID NO:34+SEQ ID NO:2+SEQ ID NO:33+SEQ ID NO:4+SEQ ID NO:5+SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8".

[0083] In one embodiment of the present invention, the circular vector 2 is composed of the following elements in the order from 5' to 3': lox66, the replication origin site ColE, tetA (tetracycline resistance gene), loxP, nox, the replication origin site oriSC101, Str (streptomycin resistance gene), loxF2, P3, P1, and P2. The nucleotide sequence of tetA (tetracycline resistance gene) is SEQ ID NO:32, and the nucleotide sequence of Str (streptomycin resistance gene) is SEQ ID NO:22. Specifically, the circular vector 2 is pSNF-2; the full sequence of pSNF-2 from the 5' end to the 3' end is "SEQ ID NO:9+SEQ ID NO:31+SEQ ID NO:32+SEQ ID NO:35+SEQ ID NO:2+SEQ ID NO:33+SEQ ID NO:22+SEQ ID NO:10+SEQ ID NO:6+SEQ ID NO:7+SEQ ID NO:8".

[0084] Further, the targeted knock-in tool 1 may consist of the following: 1) a targeting vector; the targeting vector contains a coding sequence for sgRNA targeting a specific site in the genome of the host cell; 2) a homologous recombination fragment; the homologous recombination fragment contains an upstream homologous arm, the specific DNA fragment 1, and a downstream homologous arm from the 5' to the 3' end; the upstream homologous arm is a fragment (approximately 1000 bp) located upstream of the specific site in the genome of the host cell; the downstream homologous arm is a fragment (approximately 1000 bp) located downstream of the specific site in the genome of the host cell; 3) an expression vector for expressing Cas protein and λ-red element.

[0085] In expression cassette 1, the promoter for initiating transcription of the gene encoding the Cre recombinase is ParaBAD, and the termination sequence is TrrnB. In expression cassette 2, the promoter for initiating transcription of the gene encoding the repA1 protein is Ptrc, and the termination sequence is TrrnB.

[0086] Furthermore, the specific DNA fragment 1 is composed of the following elements in a 5' to 3' order: ParaBAD, the gene encoding the Cre recombinase, TrrnB, Ptrc, the gene encoding the repA1 protein, and TrrnB.

[0087] The nucleotide sequence of ParaBAD is SEQ ID NO:16; the nucleotide sequence of the gene encoding Cre recombinase is SEQ ID NO:13; the nucleotide sequence of TrrnB is SEQ ID NO:20; the nucleotide sequence of Ptrc is SEQ ID NO:29; and the nucleotide sequence of the gene encoding repA1 protein is SEQ ID NO:30.

[0088] In one embodiment of the present invention, the specific site is located at the lpxM gene position in *E. coli*, and the knock-in involves replacing the lpxM gene (the myristyl transferase gene for lipid A biosynthesis); the coding sequence of the sgRNA is SEQ ID NO:25; the nucleotide sequence of the upstream homologous arm (lpxMup200) is SEQ ID NO:27; and the nucleotide sequence of the downstream homologous arm (lpxMdown200) is SEQ ID NO:28. In the site-specific knock-in tool 1, the targeting vector in step 1) is pTarget-lpxM; pTarget-lpxM is a recombinant plasmid obtained by replacing the sequence at positions 227-246 of plasmid pTargetF with the coding sequence of the sgRNA targeting the lpxM gene shown in SEQ ID NO:25. The expression vector used to express the Cas protein and λ-red element in step 3) is the pCAS plasmid.

[0089] This invention claims a method for constructing a targeting plasmid required for large-fragment or sequential integration of the genome of a host cell.

[0090] The method for constructing a targeting plasmid for large-fragment or sequential integration of the host cell genome, as claimed in this invention, is achieved using the kit described above; the method includes the following steps:

[0091] (c1) The specific DNA fragment 1 is knocked into a specific site in the genome of the host cell using the site-specific knock-in tool 1 (based on CRISPR-Cas9 technology).

[0092] (c2) Connect the adapter sequences to both ends of the DNA fragment to be integrated, i.e., connect one end to P1 and the other end to P2; clone the DNA fragment to be integrated into the circular vector 1 between P1 and P2 using homologous recombination to obtain recombinant vector 1; and / or clone the DNA fragment to be integrated into the circular vector 2 between P1 and P2 using homologous recombination to obtain recombinant vector 2; during the construction of recombinant vector 1 and recombinant vector 2, PCR amplification is performed using P3 as a universal primer and another specific primer for the DNA fragment to be integrated to identify whether recombinant vector 1 and recombinant vector 2 have been successfully constructed.

[0093] (c3) The recombinant vector 1 and / or the recombinant vector 2 obtained in (c2) are introduced into the host cells treated in (c1) respectively. After screening and induction of protein expression, the Cre recombinase mediates the recombination of loxF1 and loxF on the recombinant vector 1 to eliminate the replication origin site ColE on the recombinant vector 1 (leaving a loxF1 site after recombination). The resulting plasmid is the target plasmid 1 (corresponding to pKNFS2-AV1, pKNFS2-AV2, pKNFS2-AV3, pKNFS2-AV4 or pKNFS2-MVA1 in Example 6); and / or, the Cre recombinase mediates the recombination of lox66 and loxP on the recombinant vector 2 to eliminate the replication origin site ColE on the recombinant vector 2 (leaving a lox66 site after recombination). The resulting plasmid is the target plasmid 2.

[0094] The host cell may be bacteria, fungi, animal cells, or plant cells, etc.

[0095] Furthermore, the host cell may be Escherichia coli.

[0096] This invention claims protection for a kit (composition) for the large-segment or sequential integration of the genome of a host cell.

[0097] The complete product (composition) is either a complete product 3 (composition 3) for large-segment integration of the host cell genome or a complete product 4 (composition 4) for continuous large-segment integration of the host cell genome.

[0098] The complete product 3 (composition 3) includes (or is composed of): (C1), (C3) and (C4).

[0099] The complete product 4 (composition 4) includes (or is composed of): (C2), (C3) and (C4).

[0100] (C1) The aforementioned kit 1, or the target plasmid 1 or the target plasmid 2 constructed using the aforementioned method;

[0101] (C2) The aforementioned kit 2, or the target plasmid 1 and the target plasmid 2 constructed using the aforementioned method;

[0102] (C3) Circular vector 3; the circular vector B3 is used to express Nigri recombinase and Cre recombinase;

[0103] (C4) Knock-in tool 2; the knock-in tool 2 is capable of knocking in a specific DNA fragment 2 at the integration site of the host cell's genome; the specific DNA fragment 2 is composed of lox66 and nox or loxF1 and nox.

[0104] The host cell may be bacteria, fungi, animal cells, or plant cells, etc.

[0105] Furthermore, the host cell may be Escherichia coli.

[0106] Furthermore, the circular vector 3 contains an expression cassette 3 for expressing the Cre recombinase, an expression cassette 4 for expressing the Nigri recombinase, and a replication initiation site.

[0107] The amino acid sequence of the Nigri recombinase is identical to the amino acid sequence obtained by translating SEQ ID NO:15. The amino acid sequence of the Cre recombinase is identical to the amino acid sequence obtained by translating SEQ ID NO:13.

[0108] Further, in expression cassette 3, the promoter for initiating transcription of the gene encoding the Cre recombinase is ParaBAD, and the termination sequence is TrrnB. In expression cassette 4, the promoter for initiating transcription of the gene encoding the Nigri recombinase is Ptet, and the termination sequence is TrrnB. The replication initiation site is RK2s, which is a temperature-sensitive RK2 replication initiation site.

[0109] Furthermore, the circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, the resistance gene, and RK2s.

[0110] The resistance gene in the circular vector 3 is different from the resistance genes in the circular vector 1 and the circular vector 2.

[0111] The nucleotide sequence of ParaBAD is SEQ ID NO:16; the nucleotide sequence of the gene encoding Cre recombinase is SEQ ID NO:13; the nucleotide sequence of TrrnB is SEQ ID NO:20; the nucleotide sequence of Ptet is SEQ ID NO:19; the nucleotide sequence of the gene encoding Nigri recombinase is SEQ ID NO:15; and the nucleotide sequence of RK2s is SEQ ID NO:21.

[0112] In one embodiment of the present invention, the circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, Amp (ampicillin resistance gene), and RK2s. The nucleotide sequence of Amp (ampicillin resistance gene) is SEQ ID NO:23. Specifically, the circular vector 3 is pW-NC01; the full sequence of pW-NC01 from the 5' end to the 3' end is "SEQ ID NO:16+SEQ ID NO:13+SEQ ID NO:20+SEQ ID NO:19+SEQ ID NO:15+SEQ ID NO:20+SEQ ID NO:23+SEQ ID NO:21".

[0113] Further, the targeted knock-in tool 2 comprises the following: 1) a targeting vector containing a coding sequence of sgRNA targeting the integration site in the host cell's genome; 2) a homologous recombination fragment containing an upstream homologous arm, the specific DNA fragment 2, and a downstream homologous arm from the 5' to 3' end; the upstream homologous arm is a fragment (approximately 1000 bp) located upstream of the integration site in the host cell's genome; the downstream homologous arm is a fragment (approximately 1000 bp) located downstream of the integration site in the host cell's genome; 3) an expression vector for expressing the Cas protein and the λ-red element;

[0114] In one embodiment of the present invention, the host cell is *Escherichia coli*, and the integration site is located at the position of the *lpxM* gene in *E. coli*, replacing the *lpxM* gene (lipid A biosynthesis myristyltransferase gene). In the site-directed knock-in tool, the targeting vector in 1) is pTarget-lpxM; pTarget-lpxM is a recombinant plasmid obtained by replacing the sequence at positions 227-246 of plasmid pTargetF with the coding sequence of the sgRNA targeting the *lpxM* gene shown in SEQ ID NO:25. The sequence of the homologous recombination fragment in 2) is SEQ ID NO:26. The expression vector used to express the Cas protein and λ-red element in 3) is the pCAS plasmid.

[0115] This invention claims protection for a method for large-fragment or sequential integration of the genome of a host cell.

[0116] The method for large-fragment integration of the host cell genome claimed in this invention is achieved using the aforementioned product 3 (composition 3); the method may include the following steps:

[0117] (d1) Using the site-specific knock-in tool 2, the specific DNA fragment 2 is knocked into the integration site in the genome of the host cell (based on CRISPR-Cas9 technology);

[0118] (d2) The circular carrier 3 is introduced into the host cells treated with (d1);

[0119] (d3) The target targeting plasmid 1 or the target targeting plasmid 2 described above is introduced into the host cell treated in (d2), and recombinant cells with one copy of the DNA fragment to be integrated knocked into the integration site are obtained after culture and screening.

[0120] Principle: Nigri recombinase recognizes a nox-specific site and integrates the target plasmid 1 or the target plasmid 2 carrying the nox site into the pre-knocked nox site on the host cell genome. Then, Cre recombinase recognizes the pre-knocked lox66 site on the host genome and the lox71 site from the target plasmid 1, or the pre-knocked loxF1 site on the host genome and the loxF2 site from the target plasmid 2, and mediates two-site recombination, thereby deleting the replication initiation site, the resistance gene, and a nox site located between the two sites.

[0121] The method for large-fragment continuous integration of the host cell genome claimed in this invention is achieved using the aforementioned product 4 (composition 4); the method includes the following steps:

[0122] (e1) The specific DNA fragment 1 is knocked into a specific site in the genome of the host cell using the site-specific knock-in tool 1 (based on CRISPR-Cas9 technology).

[0123] (e2) Connector sequences are attached to both ends of all the DNA fragments to be integrated, i.e., one end is connected to P1 and the other end is connected to P2; after numbering the DNA fragments to be integrated, homologous recombination is used to clone the DNA fragments to be integrated with odd numbers into the circular vector 1 between P1 and P2 to obtain several recombinant vectors 1, each carrying one DNA fragment to be integrated; homologous recombination is used to clone the DNA fragments to be integrated with even numbers into the circular vector 2 between P1 and P2 to obtain several recombinant vectors 2, each carrying one DNA fragment to be integrated; during the construction of the recombinant vectors 1 and 2, PCR amplification is performed using P3 as a universal primer and another specific primer for the DNA fragment to be integrated, which can identify whether the recombinant vectors 1 and 2 have been successfully constructed.

[0124] (e3) Several recombinant vectors 1 and several recombinant vectors 2 obtained in (e2) are respectively introduced into the host cells treated in (e1). After screening and induction of protein expression, the Cre recombinase mediates the recombination of loxF1 and loxF on the recombinant vector 1 to eliminate the replication origin site ColE on the recombinant vector 1 (leaving one loxF1 site after recombination), thereby obtaining several target plasmids 1; the Cre recombinase mediates the recombination of lox66 and loxP on the recombinant vector 2 to eliminate the replication origin site ColE on the recombinant vector 2 (leaving one lox66 site after recombination), thereby obtaining several target plasmids 2.

[0125] (e4) The specific DNA fragment 2 is knocked into the integration site in the genome of the host cell using the site-specific knock-in tool 2 (based on CRISPR-Cas9 technology).

[0126] (e5) The circular carrier 3 is introduced into the host cells after (e4) treatment.

[0127] (e6) Introduce either the target plasmid 1 or the target plasmid 2 obtained in (e3) into the host cell treated in (e5), and after culturing and screening, obtain recombinant cells with the first DNA fragment to be integrated knocked into the integration site (Principle: Nigri recombinase recognizes the nox-specific site and integrates the target plasmid 1 or the target plasmid 2 carrying the nox site into the pre-knocked nox site on the host cell genome; then, Cre recombinase recognizes the pre-knocked nox site on the host genome). The lox66 site knocked into the genome and the lox71 site from the target target plasmid 1, or by recognizing the loxF1 site knocked into the host genome in advance and the loxF2 site from the target target plasmid 2, and mediating two-site recombination, thereby deleting the replication initiation site, resistance gene and a nox site located between the two sites), are denoted as recombinant cell 1; another target target plasmid 2 or the target target plasmid 1 obtained in (e3) is introduced into the recombinant cell 1, and after culture and screening, the desired integration site is obtained. The recombinant cell simultaneously knocks in one copy of the first DNA fragment to be integrated and one copy of the second DNA fragment to be integrated (Principle: Nigri recombinase recognizes a nox-specific site and integrates the target plasmid 2 or the target plasmid 1 carrying the nox site into the remaining nox site on the genome of the recombinant cell 1; then, Cre recombinase recognizes the loxF1 site retained on the recombinant cell 1 from the target plasmid 1 and the loxF2 site from the target plasmid 2). The site, or by identifying the lox66 site from the target targeting plasmid 2 and the lox71 site from the target targeting plasmid 1 retained on the recombinant cell 1, and mediating two-site recombination, thereby deleting the replication initiation site, resistance gene and a nox site located between the two sites), is denoted as recombinant cell 2; in this step, if the first introduction is the target targeting plasmid 1, then the second introduction is the target targeting plasmid 2; if the first introduction is the target targeting plasmid 2, then the second introduction is the target targeting plasmid 1.

[0128] (e7) Referring to (e6), in the alternating order of introducing the target plasmid 1 and the target plasmid 2, all the remaining target plasmid 1 and all the remaining target plasmid 2 are sequentially introduced into the recombinant cells obtained in the previous step. The final recombinant cells are the recombinant cells in which one copy of all the DNA fragments to be integrated is knocked into the integration site at the same time.

[0129] The host cell may be bacteria, fungi, animal cells, or plant cells, etc.

[0130] Furthermore, the host cell may be Escherichia coli.

[0131] The method may also include the step of eliminating the circular vector 3 (using the temperature-sensitive replication initiation site carried thereon) in the final recombinant cells by changing the culture temperature.

[0132] In this invention, the large segment is a segment of 5-55kb, such as 5.5-51kb.

[0133] This invention claims protection for any of the following applications:

[0134] (D1) The application of the aforementioned product 1 (composition 1) and / or product 2 (composition 2) in the integration or sequential integration of the genome of a host cell;

[0135] (D2) Application of the kit described above in constructing target plasmids required for large-fragment or sequential integration of the host cell genome;

[0136] (D3) The application of the aforementioned product 3 (composition 3) and / or product 4 (composition 4) in the large-segment or continuous integration of the host cell genome.

[0137] The host cell may be bacteria, fungi, animal cells, or plant cells, etc.

[0138] In one embodiment of the present invention, the host cell is Escherichia coli.

[0139] This invention claims protection for a complete set of DNA molecules.

[0140] The set of DNA molecules claimed in this invention consists of all or part of the following:

[0141] (E1) A DNA molecule with the nucleotide sequence SEQ ID NO:101 is denoted as nox4;

[0142] (E2) A DNA molecule with the nucleotide sequence SEQ ID NO:102 is denoted as nox5;

[0143] (E3) A DNA molecule with the nucleotide sequence SEQ ID NO:105 is denoted as nox8;

[0144] (E4) A DNA molecule with the nucleotide sequence SEQ ID NO:106 is denoted as nox9;

[0145] (E5) A DNA molecule with the nucleotide sequence SEQ ID NO:107 is denoted as nox10;

[0146] (E6) A DNA molecule with the nucleotide sequence SEQ ID NO:108 is denoted as nox11;

[0147] (E7) A DNA molecule with the nucleotide sequence SEQ ID NO:97 is denoted as nox.

[0148] The aforementioned part consists of two or more components.

[0149] The nox refers to the wild-type nox site sequence, while nox4, nox5, nox8, nox9, nox10, and nox11 are its variants. All seven nox sites, including the wild-type nox site, can be recognized by Nigri recombinase and recombine between two identical sites; however, each nox site is independent of the others and recombination does not occur between them.

[0150] This invention claims protection for DNA fragments or plasmids containing the set of DNA molecules described above.

[0151] The number of DNA fragments and the number of plasmids can be 1-7.

[0152] This invention claims protection for complete sets of products (compositions).

[0153] The complete product (composition) claimed in this invention may be a complete product 5 (composition 5) for simultaneous integration of multiple targets and multiple fragments into the genome of a host cell, or a complete product 6 (composition 6) for continuous simultaneous integration of multiple targets and multiple fragments into the genome of a host cell.

[0154] The complete product 5 (composition 5) may include (or be composed of): (F1), (F3) and (F4); or, the complete product 5 (composition 5) may include (or be composed of): (F2), (F3) and (F4);

[0155] The complete product 6 (composition 6) may include (or consist of) (F1), (F2), (F3) and (F4);

[0156] (F1) Several circular vectors 1; the circular vectors 1 are composed of the following elements in the order from 5' to 3': loxF1, noxM, replication origin site, resistance gene, lox71, P3, P1, P2;

[0157] (F2) Several circular vectors 2; the circular vectors 2 are composed of the following elements in the order from 5' to 3': lox66, noxM, replication origin site, resistance gene, loxF2, P3, P1, P2;

[0158] (F3) Circular vector 3; the circular vector 3 is used to express Nigri recombinase and Cre recombinase;

[0159] (F4) Several site-specific knock-in tools; the site-specific knock-in tools are capable of knocking in a specific DNA fragment at the integration site of the host cell's genome; the specific DNA fragment is composed of lox66 and noxM or loxF1 and noxM (the specific DNA fragment contains the following elements in sequence from 5' to 3': lox66 and noxM, or loxF1 and noxM);

[0160] In (F1), (F2) and (F4), noxM is none other than the nox, nox4, nox5, nox8, nox9, nox10 or nox11 mentioned above;

[0161] In different circular vectors 1, the noxM is different, and the resistance gene is different;

[0162] In different circular vectors 2, the noxM is different, and the resistance gene is different;

[0163] The noxM varies depending on the specific pinpoint input tool used.

[0164] The simultaneous integration of multiple targets and multiple fragments refers to selecting multiple (2-7) target sites on the genome of the host cell, and integrating one DNA fragment to be integrated at each target site. For each target site and each DNA fragment to be integrated, one circular vector 1 and / or one circular vector 2, as well as one site-specific knock-in tool are required.

[0165] Furthermore, for the same target point, the noxM in the annular carrier 1 and / or the annular carrier 2 is consistent with the noxM in the pinpoint knock-in tool.

[0166] P1 and P2 are adapter sequences, and the position between P1 and P2 is used to insert the DNA fragment to be integrated. P3 is a universal primer sequence used for PCR detection of the recombinant vector obtained after inserting the DNA fragment to be integrated.

[0167] The number of items is 2-7.

[0168] The integration can be either insertion or replacement.

[0169] The functions of loxF1, loxF2, lox66, and lox71 are to assist in the elimination of certain DNA fragments, such as resistance gene fragments, to facilitate the next round of integration. loxF1 and loxF2 are recognition sites for Cre recombinase; under the action of Cre recombinase, they can recombine to achieve the purpose of deleting the fragment between the two sites (the recombinant site is inactive, meaning it cannot be recognized by Cre recombinase again for recombination). lox66 and lox71 are also recognition sites for Cre recombinase; under the action of Cre recombinase, they can recombine to achieve the purpose of deleting the fragment between the two sites (the recombinant site is inactive, meaning it cannot be recognized by Cre recombinase again for recombination). Furthermore, the loxF1 / loxF2 and lox66 / lox71 recognition sites are independent of each other and will not undergo cross-recombination.

[0170] In one embodiment of the present invention, the replication initiation site is R6k in both the annular carrier 1 and the annular carrier 2.

[0171] Furthermore, in the circular vector 1 and the circular vector 2, the resistance gene is preferably different, but it can also be the same.

[0172] The resistance gene can be selected from: kanamycin resistance gene, streptomycin resistance gene, etc.

[0173] Further, the nucleotide sequence of loxF1 is SEQ ID NO:109. The nucleotide sequence of R6k is SEQ ID NO:110. The nucleotide sequence of lox71 is SEQ ID NO:111. The nucleotide sequence of P3 is SEQ ID NO:112. The nucleotide sequence of P1 is SEQ ID NO:113. The nucleotide sequence of P2 is SEQ ID NO:114. The nucleotide sequence of lox66 is SEQ ID NO:115. The nucleotide sequence of loxF2 is SEQ ID NO:116.

[0174] The amino acid sequence of the Nigri recombinase is identical to that obtained by translating SEQ ID NO:121. The amino acid sequence of the Cre recombinase is identical to that obtained by translating SEQ ID NO:118.

[0175] In one embodiment of the present invention, there are a total of 3 annular carriers 1, namely:

[0176] (1) Circular vector 1-1; the circular vector 1-1 is composed of the following elements in the order from 5' to 3': loxF1, nox, R6k, Kan (kanamycin resistance gene), lox71, P3, P1, and P2. The nucleotide sequence of the Kan (kanamycin resistance gene) is SEQ ID NO:123. Specifically, the circular vector 1-1 is pKNF-1; the full sequence of pKNF-1 from the 5' end to the 3' end is “SEQ ID NO:109+SEQ ID NO:97+SEQ ID NO:110+SEQ ID NO:123+SEQ ID NO:111+SEQ ID NO:112+SEQ ID NO:113+SEQ ID NO:114”.

[0177] (2) Circular vector 1-2; the circular vector 1-2 is composed of the following elements in the order from 5' to 3': loxF1, nox9, R6k, Str (streptomycin resistance gene), lox71, P3, P1, and P2. The nucleotide sequence of Str (streptomycin resistance gene) is SEQ ID NO:124. Specifically, the circular vector 1-2 is pSNF-9; the full sequence of pSNF-9 from the 5' end to the 3' end is "SEQ ID NO:109+SEQ ID NO:106+SEQ ID NO:110+SEQ ID NO:124+SEQ ID NO:111+SEQ ID NO:112+SEQ ID NO:113+SEQ ID NO:114".

[0178] (3) Circular vectors 1-3; the circular vectors 1-3 are composed of the following elements in the order from 5' to 3': loxF1, nox11, R6k, Cm (chloramphenicol resistance gene), lox71, P3, P1, and P2. The nucleotide sequence of Cm (chloramphenicol resistance gene) is SEQ ID NO:125. Specifically, the circular vectors 1-3 are pCNF-11; the full sequence of pCNF-11 from the 5' end to the 3' end is "SEQ ID NO:109+SEQ ID NO:108+SEQ ID NO:110+SEQ ID NO:125+SEQ ID NO:111+SEQ ID NO:112+SEQ ID NO:113+SEQ ID NO:114".

[0179] In one embodiment of the present invention, there are a total of 3 annular carriers 2, namely:

[0180] (1) Circular vector 2-1; the circular vector 2-1 is composed of the following elements in the order from 5' to 3': lox66, nox, R6k, Str (streptomycin resistance gene), loxF2, P3, P1, and P2. Specifically, the circular vector 2-1 is pSNF-1; the full sequence of pSNF-1 from the 5' end to the 3' end is "SEQ ID NO:115+SEQ ID NO:97+SEQ ID NO:110+SEQ ID NO:124+SEQ ID NO:116+SEQ ID NO:112+SEQ ID NO:113+SEQ ID NO:114".

[0181] (2) Circular vector 2-2; the circular vector 2-2 is composed of the following elements in the order from 5' to 3': lox66, nox9, R6k, Cm (chloramphenicol resistance gene), loxF2, P3, P1, and P2. Specifically, the circular vector 2-2 is pCNF-9; the full sequence of pCNF-9 from the 5' end to the 3' end is "SEQ ID NO:115+SEQ ID NO:106+SEQ ID NO:110+SEQ ID NO:125+SEQ ID NO:116+SEQ ID NO:112+SEQ ID NO:113+SEQ ID NO:114".

[0182] (3) Circular vector 2-3; the circular vector 2-3 is composed of the following elements in the order from 5' to 3': lox66, nox11, R6k, Kan (kanamycin resistance gene), loxF2, P3, P1, and P2. Specifically, the circular vector 2-3 is pKNF-11; the full sequence of pKNF-11 from the 5' end to the 3' end is "SEQ ID NO:115+SEQ ID NO:108+SEQ ID NO:110+SEQ ID NO:123+SEQ ID NO:116+SEQ ID NO:112+SEQ ID NO:113+SEQ ID NO:114".

[0183] Furthermore, the circular vector 3 contains an expression cassette 1 for expressing the Cre recombinase, an expression cassette 2 for expressing the Nigri recombinase, and a replication initiation site.

[0184] Furthermore, in expression cassette 1, the promoter for initiating transcription of the gene encoding the Cre recombinase is ParaBAD, and the termination sequence is TrrnB; and / or, in expression cassette 2, the promoter for initiating transcription of the gene encoding the Nigri recombinase is Ptet, and the termination sequence is TrrnB; and / or, the replication initiation site is RK2s, wherein RK2s is a temperature-sensitive RK2 replication initiation site.

[0185] Furthermore, the circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, the resistance gene, and RK2s.

[0186] The resistance gene in the circular vector 3 may be different from the resistance gene in the circular vector 1 and the circular vector 2.

[0187] The nucleotide sequence of ParaBAD is SEQ ID NO:117; the nucleotide sequence of the gene encoding Cre recombinase is SEQ ID NO:118; the nucleotide sequence of TrrnB is SEQ ID NO:119; the nucleotide sequence of Ptet is SEQ ID NO:120; the nucleotide sequence of the gene encoding Nigri recombinase is SEQ ID NO:121; and the nucleotide sequence of RK2s is SEQ ID NO:122.

[0188] In one embodiment of the present invention, the circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, Amp (ampicillin resistance gene), and RK2s. The nucleotide sequence of Amp (ampicillin resistance gene) is SEQ ID NO:127. Specifically, the circular vector 3 is pW-NC01; the full sequence of pW-NC01 from the 5' end to the 3' end is "SEQ ID NO:117+SEQ ID NO:118+SEQ ID NO:119+SEQ ID NO:120+SEQ ID NO:121+SEQ ID NO:119+SEQ ID NO:127+SEQ ID NO:122".

[0189] Further, the targeted knock-in tool consists of the following 1)-3): 1) a targeting vector; the targeting vector contains the coding sequence of sgRNA targeting the integration site in the genome of the host cell; 2) a homologous recombination fragment; the homologous recombination fragment contains an upstream homologous arm, the specific DNA fragment, and a downstream homologous arm from the 5' to the 3' end; the upstream homologous arm is a fragment (approximately 1000 bp) located upstream of the integration site in the genome of the host cell; the downstream homologous arm is a fragment (approximately 1000 bp) located downstream of the integration site in the genome of the host cell; 3) an expression vector for expressing Cas protein and λ-red element.

[0190] The host cell may be bacteria, fungi, animal cells, or plant cells, etc.

[0191] In one embodiment of the present invention, the host cell is *Escherichia coli*, and the integration site is all or part of the following in *E. coli*:

[0192] I) At the location of the lpxM gene, to replace the lpxM gene (lipid A biosynthesis myristyl transferase gene);

[0193] II) At the location of the poxB gene, the poxB gene (pyruvate oxidase gene) is replaced;

[0194] III) At the location of the ldhA gene, the ldhA gene (lactate dehydrogenase gene) is replaced;

[0195] IV) The pflB gene position is to replace the pflB gene (pyruvate-methanol lyase gene).

[0196] In I), the coding sequence of the sgRNA is SEQ ID NO:128 (the targeting vector is pTarget-lpxM; pTarget-lpxM is a recombinant plasmid obtained by replacing the sequence of positions 227-246 of plasmid pTargetF with the coding sequence of the sgRNA targeting the lpxM gene shown in SEQ ID NO:128), and the sequence of the homologous recombination fragment is SEQ ID NO:129.

[0197] In II), the coding sequence of the sgRNA is SEQ ID NO:130 (the targeting vector is pTarget-poxB; pTarget-poxB is a recombinant plasmid obtained by replacing the sequence of positions 227-246 of plasmid pTargetF with the coding sequence of the sgRNA targeting the poxB gene shown in SEQ ID NO:130), and the sequence of the homologous recombination fragment is SEQ ID NO:131.

[0198] In III), the coding sequence of the sgRNA is as shown in SEQ ID NO:132 (the targeting vector is pTarget-ldhA; pTarget-ldhA is a recombinant plasmid obtained by replacing the sequence of positions 227-246 of plasmid pTargetF with the coding sequence of the sgRNA targeting the ldhA gene shown in SEQ ID NO:132), and the sequence of the homologous recombination fragment is SEQ ID NO:133.

[0199] In IV), the coding sequence of the sgRNA is SEQ ID NO:134 (the targeting vector is pTarget-pflB; pTarget-pflB is a recombinant plasmid obtained by replacing the sequence of positions 227-246 of plasmid pTargetF with the coding sequence of the sgRNA targeting the pflB gene shown in SEQ ID NO:134), and the sequence of the homologous recombination fragment is SEQ ID NO:135.

[0200] In the site-directed knock-in tools described in I), II), III), and IV), the expression vector used to express the Cas protein and λ-red element described in 3) is the pCAS plasmid.

[0201] Furthermore, the targeted knock-in tool is composed of 1) and 2); and the ring carrier 3 also contains an expression cassette 3 for expressing the Cas protein and an expression cassette 4 for expressing the λ-red element.

[0202] Furthermore, in expression cassette 3, the promoter for initiating transcription of the gene encoding the Cas protein is Psal, and the termination sequence is TrrnB; and / or, in expression cassette 4, the promoter for initiating transcription of the gene encoding the λ-red element (exo-bet-gam gene) is Psal.

[0203] Furthermore, the circular vector 3 is composed of the following elements in the order from 5' to 3': Psal, the gene encoding the Cas protein, TrrnB, Psal, the exo-bet-gam gene, the TλtL3 coding sequence, the ColRNA coding sequence, ParaBAD, the gene encoding the Cre recombinase, TrrnB, Ptet, the gene encoding the Nigri recombinase, TrrnB, the resistance gene, and the RK2s.

[0204] The nucleotide sequence of Psal is SEQ ID NO:136; the encoding gene of the Cas protein is GenBank:MW718993.1, positions 3127-7233 (+) (2021-7-20); the nucleotide sequence of TrrnB is SEQ ID NO:119; the exo-bet-gam gene is GenBank:KY563785.1, positions 7320-9224 (+) (2020-4-6); the encoding sequence of TλtL3 is GenBank:MF287367.1, positions 3129-3373 (+) (2017-7-18); the encoding sequence of ColRNA is GenBank:LC377574.3, positions 6930-8489 (-) (2019-8-27); the nucleotide sequence of ParaBAD is SEQ ID NO:136. NO:117; the nucleotide sequence of the gene encoding the Cre recombinase is SEQ ID NO:118; the nucleotide sequence of the Ptet is SEQ ID NO:120; the nucleotide sequence of the gene encoding the Nigri recombinase is SEQ ID NO:121; the nucleotide sequence of the RK2s is SEQ ID NO:122.

[0205] In one embodiment of the present invention (Example 15), the circular vector 3 is composed of the following elements in the order from 5' to 3': Psal, the gene encoding the Cas protein, TrrnB, Psal, the exo-bet-gam gene, the TλtL3 coding sequence, the ColRNA coding sequence, ParaBAD, the gene encoding the Cre recombinase, TrrnB, Ptet, the gene encoding the Nigri recombinase, TrrnB, Amp (ampicillin resistance gene), and RK2s. The nucleotide sequence of the Amp (ampicillin resistance gene) is SEQ ID NO:127. Specifically, the annular carrier 3 is pW-NC02; the full sequence of pW-NC02 from the 5' end to the 3' end is “SEQ ID NO:136+GenBank:MW718993.1(3127-7233(+), 2021-7-20)+SEQ ID NO:119+SEQ ID NO:136+GenBank:KY563785.1(7320-9224(+), 2020-4-6)+GenBank:MF287367.1(3129-3373(+)2017-7-18)+GenBank:LC377574.3(6930-8489(-)2019-8-27)+SEQ ID NO:117+SEQ ID NO:118+SEQ ID NO:119+SEQ ID NO:120+SEQ ID NO:121+SEQ ID NO:119+SEQ ID NO:127+SEQ ID NO:122”.

[0206] The host cell may be a bacterium, fungus, animal cell, or plant cell;

[0207] In one embodiment of the present invention, the host cell is Escherichia coli.

[0208] This invention claims a method for simultaneous integration of multiple targets and fragments into the genome of a host cell.

[0209] The method for simultaneous integration of multiple targets and fragments into the host cell genome, as claimed in this invention, is achieved using the aforementioned product 5 (composition 5); the method may include the following steps:

[0210] (G1) The specific DNA fragment is knocked into several of the integration sites in the genome of the host cell using several of the aforementioned site-specific knock-in tools (based on CRISPR-Cas9 technology); one specific DNA fragment is knocked into each of the integration sites, and the specific DNA fragment knocked into different integration sites is different.

[0211] (G2) Connect the adapter sequences to both ends of several DNA fragments to be integrated, i.e., connect one end to P1 and the other end to P2; clone several DNA fragments to be integrated into several circular vectors 1 or several circular vectors 2 between P1 and P2 using homologous recombination to obtain several recombinant vectors; during the construction of the recombinant vectors, PCR amplification is performed using P3 as a universal primer and another specific primer for the DNA fragments to be integrated to identify whether the recombinant vector has been successfully constructed.

[0212] (G3) The circular vector 3 is introduced into the host cells treated with (G1).

[0213] (G4) The recombinant vectors obtained in (G2) are introduced into the host cells treated in (G3), and recombinant cells are obtained by culturing and screening, in which one copy of the DNA fragment to be integrated is knocked into each of the several sites to be integrated.

[0214] Principle: Nigri recombinase recognizes the noxM-specific site and integrates the recombinant vector carrying the noxM site into the host cell genome at the pre-knocked-in noxM site. Then, Cre recombinase recognizes the lox66 site pre-knocked into the host genome and the lox71 site from the circular vector 1 (in which case the specific DNA fragment carried on the site-directed knock-in tool consists of lox66 and nox), or recognizes the loxF1 site pre-knocked into the host genome and the loxF2 site from the circular vector 2 (in which case the specific DNA fragment carried on the site-directed knock-in tool consists of loxF1 and nox), and mediates two-site recombination, thereby deleting the replication origin site, the resistance gene, and one of the noxM sites located between the two sites.

[0215] The number of items is 2-7.

[0216] In one embodiment of the present invention, the method involves simultaneously or sequentially performing processes I), II), and III) described above in the genome of the host cell. This results in the simultaneous knock-in of specific DNA fragments at the lpxM, poxB, and ldhA sites of the host cell's genome. The specific DNA fragment knocked into the lpxM site contains nox, the specific DNA fragment knocked into the poxB site contains nox9, and the specific DNA fragment knocked into the ldhA site contains nox11.

[0217] Principle: Nigri recombinase recognizes specific sites for nox, nox9, and nox11. These sites on the recombinant vector 1 can recombine with their respective sites on the chromosome. Furthermore, nox, nox9, and nox11 sites do not recombine with each other. Therefore, Nigri recombinase can integrate the recombinant vector 1 carrying a specific nox (or nox mutant) site into a pre-knocked-in nox (or mutant) site with the same sequence on the host cell genome. Then, Cre recombinase recognizes the pre-knocked-in lox66 site on the host genome and the lox71 site from the recombinant vector, mediating two-site recombination, thereby deleting the replication initiation site, resistance gene, and a nox (or nox mutant) located between the two sites. The combination of lox66 and lox71 sites can be replaced by the combination of loxF1 and loxF2 sites.

[0218] This invention claims a method for the simultaneous integration of multiple targets and fragments into the genome of a host cell.

[0219] The method for simultaneous integration of multiple targets and fragments into the genome of a host cell, as claimed in this invention, is achieved using the aforementioned product 6 (composition 6); the method may include the following steps:

[0220] (H1) The specific DNA fragment is knocked into the host cell genome at N integration sites using N site-specific knock-in tools (based on CRISPR-Cas9 technology); one specific DNA fragment is knocked into each integration site, and the specific DNA fragment knocked into different integration sites is different.

[0221] (H2) Attach the adapter sequence to both ends of all the DNA fragments to be integrated, i.e., attach P1 to one end and P2 to the other end; group the DNA fragments to be integrated into groups of N, for a total of M groups, and number the groups. Using homologous recombination, clone the N DNA fragments to be integrated from the odd-numbered groups into the N circular vectors 1 between P1 and P2, respectively, to obtain several groups of recombinant vectors 1, each group containing N recombinant vectors 1, each recombinant vector 1 carrying one DNA fragment to be integrated; using homologous recombination... In the source recombination, N DNA fragments to be integrated from the even-numbered groups are cloned between P1 and P2 in N circular vectors 2, resulting in several groups of recombinant vectors 2, each containing N recombinant vectors 2, with each recombinant vector 2 carrying one DNA fragment to be integrated. During the construction of recombinant vectors 1 and 2, PCR amplification is performed using P3 as a universal primer and another specific primer for the DNA fragment to be integrated, which can identify whether the recombinant vectors 1 and 2 have been successfully constructed.

[0222] (H3) The circular carrier 3 is introduced into the host cells treated with (H1).

[0223] (H4) Any one set of recombinant vector 1 or any one set of recombinant vector 2 obtained in (H2) is introduced into the host cells treated in (H3). After culture and screening, recombinant cells are obtained in which one copy of the DNA fragment to be integrated from the first group is knocked into each of the N integration sites (Principle: Nigri recombinase recognizes the noxM specific site and integrates the recombinant vector 1 or the recombinant vector 2 carrying the noxM site into the noxM site knocked into the host cell genome in advance; then, Cre recombinase recognizes the lox66 site knocked into the host genome in advance and the lox71 site from the recombinant vector 1, or recognizes the loxF1 site knocked into the host genome in advance and the loxF2 site from the recombinant vector 2, and mediates two-site recombination, thereby deleting the replication initiation site, resistance gene and one noxM site located between the two sites), and is denoted as recombinant cell 1; then another set of recombinant vector 2 or another set of recombinant vector 1 obtained in (D2) is introduced into recombinant cell 1, and after culture and screening, recombinant cells are obtained in the first group. A recombinant cell is obtained by simultaneously knocking in one copy of the DNA fragment to be integrated from the first group and one copy of the DNA fragment to be integrated from the second group at N integration sites (principle: Nigri recombinase recognizes the noxM specific site and integrates the recombinant vector 2 or the recombinant vector 1 carrying the noxM site into the remaining noxM site on the genome of the recombinant cell 1; then, Cre recombinase recognizes the loxF1 site from the recombinant vector 1 and the loxF2 site from the recombinant vector 2 retained on the recombinant cell 1, or recognizes the lox66 site from the recombinant vector 2 and the lox71 site from the recombinant vector 1 retained on the recombinant cell 1, and mediates two-site recombination, thereby deleting the replication initiation site, the resistance gene and one noxM site located between the two sites), denoted as recombinant cell 2; in this step, if the first insertion is the recombinant vector 1, then the second insertion is the recombinant vector 2; if the first insertion is the recombinant vector 2, then the second insertion is the recombinant vector 1.

[0224] (H5) Referring to (H4), following the alternating introduction of recombinant vector 1 and recombinant vector 2, the remaining recombinant vector 1 and recombinant vector 2 of all other groups are sequentially introduced into the recombinant cells obtained in the previous step. The resulting recombinant cells are recombinant cells in which one copy of the DNA fragment to be integrated from all groups is simultaneously knocked into N integration sites. This achieves the simultaneous integration of the host cell's genome into multiple targets and fragments.

[0225] The method may also include the step of eliminating the circular vector 3 (using the temperature-sensitive replication initiation site carried thereon) in the final recombinant cells by changing the culture temperature.

[0226] N can be a natural number between 2 and 7; M can be a natural number greater than or equal to 2.

[0227] This invention claims protection for the use of the aforementioned set of DNA molecules, or the aforementioned DNA fragments or plasmids, or the aforementioned set of products 5 (composition 5), or the aforementioned set of products 6 (composition 6), in any of the following:

[0228] (I1) Simultaneous integration of multiple targets and fragments into the host cell genome;

[0229] (I2) Simultaneous integration of multiple targets and fragments into the host cell genome.

[0230] In this invention, all the aforementioned multi-target points can be 2-7 targets. Each target point is integrated into one fragment in a single integration process.

[0231] This invention claims protection for a DNA molecule.

[0232] The DNA molecule for which protection is claimed in this invention may be any of the following:

[0233] (J1) A DNA molecule with the nucleotide sequence SEQ ID NO:101 is denoted as nox4;

[0234] (J2) A DNA molecule with the nucleotide sequence SEQ ID NO:102 is denoted as nox5;

[0235] (J3) A DNA molecule with the nucleotide sequence SEQ ID NO:105 is denoted as nox8;

[0236] (J4) A DNA molecule with the nucleotide sequence SEQ ID NO:106 is denoted as nox9;

[0237] (J5) A DNA molecule with the nucleotide sequence SEQ ID NO:107 is denoted as nox10;

[0238] (J6) A DNA molecule with the nucleotide sequence SEQ ID NO:108 is denoted as nox11.

[0239] This invention claims protection for DNA fragments or plasmids containing the DNA molecules described above.

[0240] This invention claims protection for the use of the DNA molecules, DNA fragments, or plasmids described above in the integration or sequential integration of the host cell genome.

[0241] This invention claims protection for a method for integrating the genome of a eukaryotic cell.

[0242] The method for integrating exogenous DNA fragments into the genome of eukaryotic cells, as claimed in this invention, may include the following steps:

[0243] (K1) A specific DNA fragment is knocked into the integration site in the genome of a eukaryotic cell; the specific DNA fragment also contains a knock-in site-specific recombination site (hereinafter referred to as knock-in SSRS).

[0244] (K2) expresses a knock-in site-specific recombinase (hereinafter referred to as knock-in SSR) in the eukaryotic cells described in (K1);

[0245] (K3) Prepare a circular DNA molecule containing a targeting fragment; the targeting fragment includes, in order from 5' to 3', the knock-in SSRS and the DNA fragment to be integrated, consistent with those in (K1);

[0246] The knock-in SSRS can be nox, nox4, nox5, nox8, nox9, nox10 or nox11;

[0247] The knock-in SSRS can be a Nigri recombinase;

[0248] (K4) The circular DNA molecule obtained in (K3) is introduced into the eukaryotic cell described in (K2), and after culturing and screening, a copy of the DNA fragment to be integrated is knocked into the integration site in the genome of the eukaryotic cell.

[0249] Furthermore, in (K1), the specific DNA fragment may also include a knockout site-specific recombination site I (hereinafter referred to as knockout SSRS-I), which is located at the 5' end (upstream) of the knock-in SSRS. In some embodiments of the present invention, the specific DNA fragment is composed of the knockout SSRS-I and the knock-in SSRS from the 5' end to the 3' end.

[0250] Furthermore, in the (K2) formulation, a knockout site-specific recombinase (hereinafter referred to as knockout SSR) is also simultaneously expressed in the eukaryotic cells.

[0251] Furthermore, in (K3), the target fragment includes, in order from 5' to 3', the knock-in SSRS, the knock-out site-specific recombination site II (hereinafter referred to as knock-out SSRS-II), and the DNA fragment to be integrated, which are consistent with those in (K1).

[0252] Further, the knockout SSRS-I and the knockout SSRS-II are 10x66 and 10x71 respectively; or, the knockout SSRS-I and the knockout SSRS-II are 10xF1 and 10xF2 respectively.

[0253] Furthermore, the knocked-out SSR is a Cre recombinase.

[0254] The integration can be either insertion or replacement.

[0255] The loxF1 and loxF2 are recognition sites of Cre recombinase. Under the action of Cre recombinase, they can recombine to achieve the purpose of deleting the segment between the two sites (after recombination, the site on the genome is inactive as loxF3, that is, it cannot be recognized by Cre recombinase again to recombine). The lox66 and lox71 are also recognition sites of Cre recombinase. Under the action of Cre recombinase, they can recombine to achieve the purpose of deleting the segment between the two sites (after recombination, the site on the genome is inactive as lox72, that is, it cannot be recognized by Cre recombinase again to recombine). Furthermore, the two pairs of recognition sites, loxF1 / loxF2 and lox66 / lox71, are independent of each other and will not undergo cross-recombination.

[0256] The Nigri recombinase can be replaced with a polypeptide having Nigri recombinase activity. The Cre recombinase can be replaced with a polypeptide having Cre recombinase activity. The substrate recognized by the Nigri recombinase is the nox sequence or its mutant. The substrate recognized by the Cre recombinase is loxP or its mutant, such as loxF1, loxF2, lox66, or lox71.

[0257] Optionally, the circular DNA molecule may also contain a selection gene. The selection gene may include resistance genes that are genetically manipulated in E. coli, such as ampicillin, kanamycin, or streptomycin resistance genes; or selection genes that are selected in recipient eukaryotic cells, such as G418 resistance genes, hygromycin resistance genes, or auxotrophic genes such as URA3 or Leu.

[0258] Alternatively, the upstream of the knockout SSRS-II segment may also contain a prokaryotic replication origin (such as ColE).

[0259] In some embodiments of the present invention, the specific DNA fragment consists of lox66 and nox from the 5' end to the 3' end. Correspondingly, the targeting fragment consists of nox, URA3, ColE, Amp, lox71, the DNA fragment to be integrated, and loxF1 from the 5' end to the 3' end. Principle: Nigri recombinase recognizes the nox-specific site and integrates the targeting fragment carrying the nox site into the pre-knock-in nox site on the eukaryotic genome; then, Cre recombinase recognizes the pre-knock-in lox66 site on the eukaryotic genome and the lox71 site from the targeting fragment, and mediates two-site recombination, thereby deleting a nox site located between the two sites, as well as URA3, ColE, and Amp.

[0260] In other embodiments of the invention, the specific DNA fragment consists of lox66 and nox from the 5' end to the 3' end. Correspondingly, the targeting fragment consists of nox, a Cre recombinase expression cassette, ColE, Amp, lox71, the DNA fragment to be integrated, and loxF1 from the 5' end to the 3' end. After the Nigri recombinase recognizes the nox-specific site and integrates the targeting fragment into the nox site, the integrated Cre recombinase functions to achieve recombination at the lox66 and lox71 sites.

[0261] Furthermore, in step (K1), the specific DNA fragment to be integrated into the genome of the eukaryotic cell can be achieved using a site-directed knock-in tool.

[0262] In some embodiments of the present invention, the site-directed knock-in tool may be a CRISPR-Cas gene editing tool, i.e., the step (K1) is completed based on CRISPR-Cas technology. Specifically, the site-directed knock-in tool consists of a vector capable of expressing sgRNA and Cas proteins (such as Cas9, Ca12a, dCas9, etc.) targeting the integration site, and a homologous recombination fragment; the homologous recombination fragment consists of an upstream homologous arm, the specific DNA fragment, and a downstream homologous arm; the upstream and downstream homologous arms are sequences in the eukaryotic cell genome located upstream and downstream of the integration site, respectively.

[0263] In other embodiments of the present invention, the site-specific knock-in tool may be a prime editing tool, i.e., a CRISPR-Cas nickase / reverse transcriptase-mediated technique to complete the step (K1). Specifically, the site-specific knock-in tool consists of a pegRNA or twinPE pegRNA molecule or expression vector into which a specific sequence is inserted to the integration site, and an RNA molecule or expression vector expressing a Cas nickase (such as Cas9 nickase (H840A)) and a reverse transcriptase.

[0264] Furthermore, the pinpoint knock-in tool described in this invention can also be any other similar tool with the same function, such as transposases or CRISPR-Cas / transposon editing systems.

[0265] Further, in step (K2), the expression of Nigri recombinase and Cre recombinase in the eukaryotic cells of (K1) can be achieved by introducing vectors capable of expressing Nigri and Cre recombinase into the eukaryotic cells. This can be achieved by introducing the coding genes for Nigri and Cre recombinase into a single vector, or by introducing two vectors (each carrying the coding gene for Nigri and the coding gene for Cre recombinase), or by introducing Nigri recombinase in the form of a vector while Cre recombinase is located within the circular DNA molecule.

[0266] This invention claims a method for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells.

[0267] The method for sequential integration of exogenous DNA fragments into the genome of eukaryotic cells, as claimed in this invention, may include the following steps:

[0268] (L1) A specific DNA fragment is knocked into the integration site in the genome of a eukaryotic cell, the specific DNA fragment consisting of knock-out SSRS-A and knock-in SSRS from the 5' end to the 3' end;

[0269] (L2) Knock-in SSR and knock-out SSR are expressed in the eukaryotic cells after (L1) treatment;

[0270] (L3) Prepare several circular DNA molecules; each circular DNA molecule contains a targeting fragment, and each targeting fragment contains a DNA fragment to be integrated; the several circular DNA molecules are divided into the following two categories according to the structural differences of the targeting fragments they contain:

[0271] Category 1: Several circular DNA molecules 1 carrying a type I structure targeting fragment; the type I structure targeting fragment includes, in order from 5' to 3', the knock-in SSRS, knock-out SSRS-B, DNA fragment to be integrated, and knock-out SSRS-C, consistent with those in (L1);

[0272] Category 2: Several circular DNA molecules 2 carrying a type II structure targeting fragment; the type II structure targeting fragment includes, in order from 5' to 3', the knock-in SSRS, the knock-out SSRS-D, the DNA fragment to be integrated, and the knock-out SSRS-A, which are consistent with those in (L1);

[0273] The knockout SSRS-A, knockout SSRS-B, knockout SSRS-C, and knockout SSRS-D can be 10x66, 10x71, 10xF1, and 10xF2, respectively; or, the knockout SSRS-A, knockout SSRS-B, knockout SSRS-C, and knockout SSRS-D can be 10xF1, 10xF2, 10x66, and 10x71, respectively.

[0274] The knock-in SSRS can be nox, nox4, nox5, nox8, nox9, nox10 or nox11;

[0275] The knock-in SSR can be Nigri recombinase; the knock-out SSR can be Cre recombinase.

[0276] (L4) Introduce any one of the circular DNA molecules 1 obtained in (L3) into the eukaryotic cells of (L2), and after culturing and screening, obtain a recombinant cell with one copy of the first DNA fragment to be integrated knocked into the integration site, denoted as recombinant cell 1; then introduce the other circular DNA molecule 2 obtained in (L3) into the recombinant cell 1, and after culturing and screening, obtain a recombinant cell with one copy of the first DNA fragment to be integrated and one copy of the second DNA fragment to be integrated knocked into the integration site, denoted as recombinant cell 2;

[0277] (L5) Referring to (L4), in the alternating order of introducing the circular DNA molecule 1 and the circular DNA molecule 2, all the remaining circular DNA molecules 1 and all the remaining circular DNA molecules 2 are sequentially introduced into the recombinant cells obtained in the previous step, that is, to achieve the insertion of one copy of all the DNA fragments to be integrated or the selective insertion of the DNA fragments of interest into the site to be integrated.

[0278] The integration can be either insertion or replacement.

[0279] The Nigri recombinase can be replaced with a polypeptide having Nigri recombinase activity. The Cre recombinase can be replaced with a polypeptide having Cre recombinase activity. The substrate recognized by the Nigri recombinase is the nox sequence or its mutant. The substrate recognized by the Cre recombinase is loxP or its mutant, such as loxF1, loxF2, lox66, or lox71.

[0280] Optionally, the circular DNA molecule may also contain a selection gene. The selection gene may include resistance genes that are genetically manipulated in E. coli, such as ampicillin, kanamycin, or streptomycin resistance genes; or selection genes that are selected in recipient eukaryotic cells, such as G418 resistance genes, hygromycin resistance genes, or auxotrophic genes such as URA3 or Leu.

[0281] Alternatively, in the target segment, the upstream of the knockout SSRS-C and / or the knockout SSRS-D may also contain a prokaryotic replication origin (such as ColE).

[0282] In some embodiments of the present invention, the specific DNA fragment consists of lox66 and nox from the 5' end to the 3' end. Correspondingly, the targeting fragment consists of nox, URA3, ColE, Amp, lox71, the DNA fragment to be integrated, and loxF1 from the 5' end to the 3' end.

[0283] In other embodiments of the invention, the specific DNA fragment consists of lox66 and nox from the 5' end to the 3' end. Correspondingly, the targeting fragment consists of nox, Cre recombinase expression cassette, ColE, Amp, lox71, the DNA fragment to be integrated, and loxF1 from the 5' end to the 3' end.

[0284] Furthermore, in step (L1), the specific DNA fragment to be knocked into the integration site in the genome of the eukaryotic cell can be achieved using a site-directed knock-in tool.

[0285] In some embodiments of the present invention, the site-directed knock-in tool may be a CRISPR-Cas gene editing tool, i.e., the step (L1) is completed based on CRISPR-Cas technology. Specifically, the site-directed knock-in tool consists of a vector capable of expressing sgRNA and Cas proteins (such as Cas9, Ca12a, dCas9, etc.) targeting the integration site, and a homologous recombination fragment; the homologous recombination fragment consists of an upstream homologous arm, the specific DNA fragment, and a downstream homologous arm; the upstream and downstream homologous arms are sequences in the eukaryotic cell genome located upstream and downstream of the integration site, respectively.

[0286] In other embodiments of the present invention, the site-specific knock-in tool may be a prime editing tool, i.e., a technology that completes the step (L1) based on Cas nickase / reverse transcriptase-mediated technology. Specifically, the site-specific knock-in tool consists of a pegRNA or twinPE pegRNA molecule or expression vector into which a specific sequence is inserted to the integration site, and an RNA molecule or expression vector expressing a Cas nickase (such as Cas9 nickase (H840A)) and a reverse transcriptase.

[0287] Further, in step (L2), the expression of Nigri recombinase and Cre recombinase in the eukaryotic cells of (L1) can be achieved by introducing vectors capable of expressing Nigri and Cre recombinase into the eukaryotic cells. This can be achieved by introducing the encoding genes for Nigri and Cre recombinase into a single vector, or by introducing two vectors (each carrying the encoding genes for Nigri and Cre recombinase respectively), or by introducing Nigri recombinase into a vector while Cre recombinase is located within the circular DNA molecule.

[0288] This invention claims protection for a method for library integration of the genome of eukaryotic cells.

[0289] The method for integrating a library of a eukaryotic cell genome claimed in this invention includes the steps (K1)-(K4) above, wherein the DNA fragment to be integrated is a library sequence;

[0290] The library sequence is a collection of DNA fragments containing multiple independent target sequences, and each independent target sequence is integrated into the genome of a eukaryotic cell through steps (K1)-(K4) to obtain multiple integrated cell lines containing different target sequences.

[0291] This invention claims protection for a method for multi-site integration of the genome of a eukaryotic cell.

[0292] The method for multi-site integration of the genome of a eukaryotic cell, as claimed in this invention, may include the following steps:

[0293] (M1) Select multiple different integration sites in the genome of a eukaryotic cell and knock in a specific DNA fragment into each integration site; wherein the specific DNA fragments knocked in at different integration sites are different from each other;

[0294] The specific DNA fragment consists of knockout SSRS-I and knock-in SSRS from the 5' end to the 3' end;

[0295] (M2) expresses knock-in SSR and knock-out SSR in the eukaryotic cells of (M1);

[0296] (M3) Prepare circular DNA molecules containing the targeting fragment for each integration site to obtain several circular DNA molecules; the targeting fragment includes the knock-in SSRS, knock-out SSRS-II, and DNA fragment to be integrated in the order of 5' to 3'; wherein, for each integration site, the knock-in SSRS in the targeting fragment is the same as the knock-in SSRS knocked in at that site in step (M1);

[0297] The knockout SSRS-I and the knockout SSRS-II can be 10x66 and 10x71 respectively; or, the knockout SSRS-I and the knockout SSRS-II can be 10xF1 and 10xF2 respectively.

[0298] The knock-in SSRS can be nox, nox4, nox5, nox8, nox9, nox10 or nox11, and the knock-in SSRS in any two different specific DNA fragments in (M1) are different from each other;

[0299] The knock-in SSR can be Nigri recombinase; the knock-out SSR can be Cre recombinase.

[0300] (M4) Several circular DNA molecules obtained in (M3) are introduced into the eukaryotic cell of (M2), and after culturing and screening, the DNA fragments to be integrated are knocked into different integration sites in the genome of the eukaryotic cell.

[0301] In this method, the DNA fragments to be integrated can be different or the same, knocked into different sites.

[0302] The integration can be either insertion or replacement.

[0303] In this context, Nigri recombinase can be replaced with a polypeptide having Nigri recombinase activity. Cre recombinase can be replaced with a polypeptide having Cre recombinase activity.

[0304] In some embodiments of the present invention, there are three integration sites, and the three specific DNA fragments are respectively referred to as specific DNA fragment 1, specific DNA fragment 2 and specific DNA fragment 3; specific DNA fragment 1 consists of lox66 and nox from the 5' end to the 3' end, specific DNA fragment 2 consists of lox66 and nox4 from the 5' end to the 3' end, and specific DNA fragment 3 consists of lox66 and nox5 from the 5' end to the 3' end. The three corresponding targeting fragments are respectively designated as targeting fragment 1, targeting fragment 2, and targeting fragment 3. Target fragment 1 consists of nox, URA3, ColE, Amp, lox71, the DNA fragment to be integrated, and loxF1 from the 5' end to the 3' end. Target fragment 2 consists of nox4, LEU, ColE, Amp, lox71, the DNA fragment to be integrated, and loxF1 from the 5' end to the 3' end. Target fragment 3 consists of nox5, NTC, ColE, Amp, lox71, the DNA fragment to be integrated, and loxF1 from the 5' end to the 3' end.

[0305] Furthermore, in step (M1), knocking in a specific DNA fragment to each of the sites to be integrated can be achieved using a site-specific knock-in tool.

[0306] In some embodiments of the present invention, the site-directed knock-in tool is a CRISPR-Cas gene editing tool, that is, step (M1) is completed based on CRISPR-Cas technology. Specifically, the site-directed knock-in tool consists of a vector capable of expressing sgRNA and Cas proteins (such as Cas9, Ca12a, dCas9, etc.) targeting the integration site, and a homologous recombination fragment; the homologous recombination fragment consists of an upstream homologous arm, the specific DNA fragment, and a downstream homologous arm; the upstream and downstream homologous arms are sequences in the eukaryotic cell genome located upstream and downstream of the integration site, respectively.

[0307] Further, in step (M2), the expression of Nigri recombinase and Cre recombinase in the eukaryotic cells treated in (M1) can be achieved by introducing vectors capable of expressing Nigri and Cre recombinase into the eukaryotic cells. This can be achieved by introducing the coding genes for Nigri and Cre recombinase into a single vector, or by introducing two vectors (each carrying the coding genes for Nigri and Cre recombinase respectively), or by introducing Nigri recombinase into a vector while Cre recombinase is located within the circular DNA molecule.

[0308] In this invention, the multiple sites can be 2-7 sites. In some embodiments of this invention, the multiple sites are 3 sites.

[0309] This invention claims protection for a kit (composition) for integrating exogenous DNA fragments into the genome of eukaryotic cells.

[0310] The kit product (referred to as kit product 7 or composition 7) for integrating exogenous DNA fragments into the genome of eukaryotic cells claimed in this invention includes at least Nigri recombinase or a polypeptide having Nigri recombinase activity.

[0311] The recognition site of the recombinase Nigri is nox or a mutant thereof. Nox or a mutant thereof is knocked into the chromosome of a eukaryotic cell beforehand, and under the action of the recombinase Nigri, it can recombine with nox or a mutant thereof located on the target fragment sequence with the same sequence, thereby mediating the efficient integration of the target fragment sequence into the chromosome.

[0312] The complete product 7 (composition 7) also includes recombinase Cre or a polypeptide having recombinase Cre activity.

[0313] The recognition site of the recombinase Cre is a loxP sequence or a mutant thereof. Paired loxP sequences or mutants thereof located at both ends of a specific gene segment on the chromosome can recombine under the action of the recombinase Cre, thereby knocking out the specific gene segment from the chromosome.

[0314] Optionally, the product 7 (composition 7) may further include a protein with CAS activity, which is capable of substrate recognition at specific sequences on eukaryotic chromosomes mediated by sgRNA. More selectively, the protein with CAS activity includes Cas9, Ca12a, dCas9, etc. More selectively, the protein with CAS activity can be mutated to retain only nickase activity and fused with a sequence having reverse transcriptase activity, and under the action of pegRNA, induce effector responses including leader editing, one result of which is the insertion of nox or its mutant, loxP or its mutant, or combinations thereof, at specific locations on eukaryotic chromosomes. The product 7 (composition 7) can efficiently integrate large fragments at any location on eukaryotic chromosomes. The integration site is determined by the pegRNA sequence.

[0315] The kit product (i.e., kit product 7 or composition 7) claimed in this invention for integrating exogenous DNA fragments into the genome of eukaryotic cells may include:

[0316] (f1) Site-specific knock-in tool; the site-specific knock-in tool is capable of knocking in the specific DNA fragment described in step (K1) above into the integration site in the genome of a eukaryotic cell;

[0317] (f2) A recombinase or a nucleic acid molecule capable of expressing the recombinase or an expression cassette or recombinant plasmid containing the nucleic acid molecule; the recombinase is Nigri recombinase; or the recombinase is a combination of Nigri recombinase and Cre recombinase;

[0318] (f3) Specific vector; the specific vector is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule described in step (K3) above. The specific vector is used to insert the desired DNA fragment to be integrated to construct a new recombinant vector.

[0319] In some embodiments of the present invention, the site-directed knock-in tool is a CRISPR-Cas9 gene editing tool. Specifically, the site-directed knock-in tool consists of a vector capable of expressing sgRNA and Cas9 protein targeting the integration site, and a homologous recombination fragment; the homologous recombination fragment consists of an upstream homologous arm, the specific DNA fragment, and a downstream homologous arm; the upstream and downstream homologous arms are sequences in the eukaryotic cell genome located upstream and downstream of the integration site, respectively.

[0320] In other embodiments of the present invention, the site-specific knock-in tool may be a prime editing tool, i.e., a CRISPR-Cas nickase / reverse transcriptase-mediated technique to complete step (B1). Specifically, the site-specific knock-in tool consists of a pegRNA or twinPE pegRNA molecule or expression vector into which a specific sequence is inserted to the integration site, and an RNA molecule or expression vector expressing a Cas nickase (such as Cas9 nickase (H840A)) and a reverse transcriptase.

[0321] This invention claims protection for a complete product (composition) for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells.

[0322] The kit product (referred to as kit product 8 or composition 8) for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells claimed in this invention includes at least Nigri recombinase or a polypeptide having Nigri recombinase activity.

[0323] The recognition site of the recombinase Nigri is nox or a mutant thereof. Nox or a mutant thereof is knocked into the chromosome of a eukaryotic cell beforehand, and under the action of the recombinase Nigri, it can recombine with nox or a mutant thereof located on the target fragment sequence with the same sequence, thereby mediating the efficient integration of the target fragment sequence into the chromosome.

[0324] The complete product 8 (composition 8) also includes recombinase Cre or a polypeptide having recombinase Cre activity.

[0325] The recognition site of the recombinase Cre is a loxP sequence or a mutant thereof. Paired loxP sequences or mutants thereof located at both ends of a specific gene segment on the chromosome can recombine under the action of the recombinase Cre, thereby knocking out the specific gene segment from the chromosome.

[0326] Furthermore, the complete product 8 (composition 8) also includes a vector carrying a targeting fragment. The targeting fragment includes a DNA fragment sequentially linked by noxM-exogenous gene insertion site-lox(B), and the targeting fragment is a circular DNA sequence; simultaneously, the complete product 8 (composition 8) also includes a sequence capable of inserting lox(A)-noxM onto a eukaryotic chromosome.

[0327] Furthermore, the noxM sequence can be the nox sequence or its mutants. In a single operation, the noxM sequence on the chromosome is completely identical to the noxM sequence of the targeted fragment.

[0328] Furthermore, the lox(A) sequence can be the loxP sequence or its mutants. During a single operation, the lox(A) sequence on the chromosome appears in pairs with the lox(B) sequence of the target fragment, maintaining a fixed pairing. Further, the aforementioned fixed lox(A) / lox(B) sequence combinations can be: lox66 / lox71, loxF1 / loxF2. Furthermore, other lox(A) / lox(B) sequence combinations with the same function can also be used.

[0329] Optionally, the product 8 (composition 8) may further include a protein with CAS activity, which is capable of substrate recognition at specific sequences on eukaryotic chromosomes mediated by sgRNA. More selectively, the protein with CAS activity includes Cas9, Ca12a, dCas9, etc. More selectively, the protein with CAS activity can be mutated to retain only nickase activity and fused with a sequence having reverse transcriptase activity, and under the action of pegRNA, induce effector responses including leader editing, one result of which is the insertion of nox or its mutant, loxP or its mutant, or combinations thereof, at specific locations on eukaryotic chromosomes. The product 8 (composition 8) can efficiently integrate large fragments at any location on eukaryotic chromosomes. The integration site is determined by the pegRNA sequence.

[0330] The kit product (i.e., kit product 8 or composition 8) claimed in this invention for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells may include:

[0331] (g1) Site knock-in tool; the site knock-in tool is capable of knocking in the specific DNA fragment described in step (L1) above into the integration site in the genome of a eukaryotic cell;

[0332] (g2) Nigri recombinase and Cre recombinase; or a nucleic acid molecule capable of expressing Nigri recombinase and Cre recombinase or an expression cassette or recombinant plasmid containing said nucleic acid molecule;

[0333] (g3) Specific vector 1 and specific vector 2; the specific vector 1 is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule 1 described in step (L3) above; the specific vector 2 is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule 2 described in step (L3) above. The specific vector 1 and the specific vector 2 are used to insert the desired DNA fragment to be integrated to construct new circular DNA molecule 1 and circular DNA molecule 2.

[0334] This invention claims protection for a kit (composition) for multi-site integration of the genome of eukaryotic cells.

[0335] The kit for multi-site integration of the genome of eukaryotic cells claimed in this invention (referred to as kit 9 ​​or composition 9) may include:

[0336] (h1) Fixed-point typing tool; the fixed-point typing tool can perform the steps (M1) described above;

[0337] (h2) Nigri recombinase and Cre recombinase; or a nucleic acid molecule capable of expressing Nigri recombinase and Cre recombinase or an expression cassette or recombinant plasmid containing said nucleic acid molecule;

[0338] (h3) Several specific vectors; the several specific vectors are obtained by deleting only the DNA fragment to be integrated from the several circular DNA molecules described in step (M3) above (the specific vectors correspond one-to-one with the circular DNA molecules). The several specific vectors are used to insert the required DNA fragment to be integrated to construct several new circular DNA molecules.

[0339] In this invention, all the recombinases Cre and their recognition sequences described above can be replaced by recombinases such as FLP, Dre, and Panta and their recognition sequences.

[0340] In this invention, all the DNA fragments to be integrated mentioned above are fragments of 60kb or less.

[0341] In this invention, the nucleotide sequences of all the aforementioned nox are as shown in SEQ ID NO:182; the nucleotide sequence of nox4 is as shown in SEQ ID NO:183; the nucleotide sequence of nox5 is as shown in SEQ ID NO:184; the nucleotide sequence of nox8 is as shown in SEQ ID NO:185; the nucleotide sequence of nox9 is as shown in SEQ ID NO:186; the nucleotide sequence of nox10 is as shown in SEQ ID NO:187; and the nucleotide sequence of nox11 is as shown in SEQ ID NO:188.

[0342] In this invention, the nucleotide sequences of all lox66 described above are shown in SEQ ID NO:189; the nucleotide sequence of lox71 is shown in SEQ ID NO:190; the nucleotide sequence of loxF1 is shown in SEQ ID NO:191; and the nucleotide sequence of loxF2 is shown in SEQ ID NO:192.

[0343] This invention claims protection for the use of the aforementioned complete product 7 (composition 7), complete product 8 (composition 8), or complete product 9 (composition 9) in any of the following:

[0344] P1. Editing animal cell chromosomes;

[0345] P2. Editing plant cell chromosomes;

[0346] P3. Editing fungal cell chromosomes.

[0347] The fungal cells may include, but are not limited to: Yersinia lipolytica, Pichia pastoris, Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces var. kanamycin, Trichoderma, and Aspergillus.

[0348] The animal cells may include, but are not limited to, cells derived from: humans, mice, rats, monkeys, dogs, pigs, sheep, cattle, cats, chickens, ducks, geese, etc. Specifically, they may be 293T cells, CHO cells, or primary cells.

[0349] The plant cells may include, but are not limited to, monocotyledonous and dicotyledonous plants, specifically rice, corn, wheat, sorghum, barley, soybean, peanut, Arabidopsis thaliana, etc.

[0350] This invention claims protection for the use of the aforementioned product 7 (composition 7), the aforementioned product 8 (composition 8), the aforementioned product 9 (composition 9), or the aforementioned method in any of the following:

[0351] Q1. Modification of eukaryotic microbial engineered strains;

[0352] Q2. Construction of stable mammalian cell lines;

[0353] Q3. Crop plant modification.

[0354] In some embodiments of the present invention, the modification of the eukaryotic microbial engineered strain specifically refers to the modification of the chassis cells of Yersinia lipophila.

[0355] In some embodiments of the present invention, the construction of the stable mammalian cell line specifically involves constructing a mutant library stably expressing CD19 using CHO cells. In some embodiments of the present invention, the construction of the stable mammalian cell line specifically involves constructing a CAR-T stable expression cell line.

[0356] In some embodiments of the present invention, the crop plant modification specifically refers to the modification of the stress resistance traits of corn plants. Attached Figure Description

[0357] Figure 1 is a schematic diagram of the process of integrating large continuous gene fragments in eukaryotic cells using this method.

[0358] Figure 2 shows the results of multiple rounds of integration of the *Yarrowia lipophila* genome in Example 21. PN represents the number of positive clones; PR represents the positive integration rate. Figures 1-3 represent the first, second, and third rounds of integration experiments, respectively.

[0359] Figure 3 shows the primer designs for gene editing detection in CHO cells in Examples 24 and 25. (A) shows the primer designs for identifying the EF1α promoter library (protein expression library) in Example 24; (B) shows the primer designs for identifying the CD19 mutant library in Example 25.

[0360] Figure 4 shows the positive integration efficiency of CHO cells in Examples 24 and 25. "E" indicates the integration rate of the EF1α promoter library in Example 9; "C" indicates the integration rate of the CD19 mutant library in Example 10. PI% represents the percentage of different cell types identified by PCR (1: integration-positive cells; 2: integration-negative cells; 3: heterozygous cells; 4: incompletely integrated cells; 5: randomly integrated cells; 6: cells of unclear identification). Embodiments of the present invention

[0361] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0362] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0363] Example 1: Construction of strains BN-01, BN-02, BN-03, BN-04, and BN-05

[0364] The CRISPR-Cas9 method follows the procedure described in the literature "Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514." The integration recognition site "lox66-nox" is integrated into the myristoyltransferase gene lpxM in the chromosome of *E. coli* strain BW25113 (lox66 first, nox second in 5'-3' order), and the lpxM gene is then knocked out. The specific procedures are as follows:

[0365] 1-1) Using the genome of Escherichia coli BW25113 as a template, homologous arm fragments L1 and L2, each 1000 bp in length upstream and downstream of the integration site gene, were amplified by PCR using primers lpxM-up-F / lpxM-up-R and lpxM-down-F / lpxM-down-R (Table 1) (the primers contain the lox66-nox site).

[0366] 1-2) Using L1 and L2 obtained in 1-1) as templates, and lpxM-up-F / lpxM-down-R as primers, a complete gene fragment was obtained by PCR amplification, denoted as L3.

[0367] The L3 sequence is as follows:

[0368]

[0369] 1-3) Design primers lpxM-N20-F / lpxM-N20-R, and use plasmid pTargetF (refer to the literature "Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514.") as a template for PCR amplification. After the PCR product is treated with DpnI enzyme, it is transformed into DH5α to obtain plasmid pTarget-lpxM.

[0370] The structure of plasmid pTarget-lpxM is achieved by replacing the sequence from position 227 to 246 of plasmid pTargetF (described in “Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015; 81(7):2506-14.”, which is available to the public from the applicant) with sequence 5'-ATGATCATACGATTGCGCGG-3' (the coding sequence of the sgRNA targeting the lpxM gene, SEQ ID NO:25).

[0371] 1-4) Prepare Escherichia coli BW25113 electroporation competent cells, and transfer the pCAS plasmid (described in “Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015; 81(7):2506-14.”, which is available to the public from the applicant) into the cells by electroporation. Culture the cells on kanamycin plates at 30°C. Select single clones and designate them as BW25113 / Pcas strain.

[0372] 1-5) The obtained BW25113 / Pcas strain was inoculated into LB medium containing 50 mg / L kanamycin, and then 0.2% L-arabinose was added to the medium. The cells were cultured at 30°C for 3-4 hours to prepare electrotransformation competent cells.

[0373] 1-6) The target fragment L3 (SEQ ID NO:26) and plasmid pTarget-lpxM obtained above were co-transformed into the electrotransformation competent cells obtained in step 1-5). After culturing at 30°C for 1 hour, the cells were spread on solid LB culture dishes containing 50 mg / L kanamycin and 50 mg / L streptomycin and placed in an incubator at 30°C until single clones grew.

[0374] 1-7) Perform colony PCR verification on the single colonies obtained above. The verification primers are lpxM-up-F284 / lpxM-down-R195. If the band size is only about 2000bp, it means that lox66-nox has been successfully integrated at the lpxM site. Pick the correct single colonies and incubate them overnight at 30°C in liquid LB medium containing 50mg / L kanamycin and 0.5mM IPTG.

[0375] 1-8) The overnight cultured bacterial suspension was streaked onto solid LB medium containing 50 mg / L kanamycin and incubated at 30°C. After single colonies grew, single colonies were picked and placed on solid LB medium containing 50 mg / L kanamycin and 50 mg / L kanamycin and streptomycin, respectively, and then incubated at 30°C. If the inoculated single colonies grew on solid LB medium containing 50 mg / L kanamycin but not on solid LB medium containing 50 mg / L streptomycin, then the pTarget-lpxM plasmid elimination was completed (the principle and process are described in the literature "Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514.").

[0376] 1-9) Single colonies with the pTarget-lpxM plasmid eliminated were picked and cultured overnight at 42°C on liquid LB medium. Then, they were streaked onto antibiotic-free solid LB medium and cultured at 37°C for 12 hours. After single colonies grew, they were picked and inoculated onto antibiotic-free and solid LB medium containing 50 mg / L kanamycin, respectively, and incubated overnight at 37°C. If no growth was observed on solid LB medium containing 50 mg / L kanamycin, then the pCAS plasmid elimination was successful (the principle and process are described in the literature "Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514."). The obtained single colonies are strain BN-01.

[0377] The BN-01 strain is obtained by replacing the lpxM gene in the genome of Escherichia coli BW25113 with the integration recognition site "lox66-nox" while keeping other sequences unchanged.

[0378] 1-10) Following the procedures in 1-1) to 1-9), starting with strain BW25113, BN-02, BN-03, BN-04, and BN-05 were obtained respectively. The difference is that in the process of constructing strain BN-0 "N", the primers used were replaced with the corresponding primers [number 01A-H] in Table 1 with primers [number 0 "N"AH]. The BN-02 strain is obtained by replacing the lpxL gene in the genome of Escherichia coli BW25113 with the integration recognition site “lox66-nox”, while keeping other sequences unchanged; the BN-03 strain is obtained by replacing the poxB gene in the genome of Escherichia coli BW25113 with the integration recognition site “lox66-nox”, while keeping other sequences unchanged; the BN-04 strain is obtained by replacing the pflB gene in the genome of Escherichia coli BW25113 with the integration recognition site “lox66-nox”, while keeping other sequences unchanged; the BN-05 strain is obtained by replacing the ldhA gene in the genome of Escherichia coli BW25113 with the integration recognition site “lox66-nox”, while keeping other sequences unchanged.

[0379] In this application, strains BN-02, BN-03, BN-04, and BN-05 are replacement strains for BN-01, and are used in exactly the same way.

[0380] Table 1. Primer sequences involved in this embodiment

[0381] Example 2: Construction of tool plasmids

[0382] All the tool plasmids used were prepared by whole-gene synthesis or splicing methods. The relevant information is shown in Tables 2 and 3.

[0383] Table 2. Plasmid Information Note: loxF1 and loxF2 are a pair of recognition sites for Cre recombinase. Under the action of Cre recombinase, they can recombine (the new site formed after recombination is inactive, meaning it cannot be recognized by Cre recombinase to undergo recombination), thus achieving the purpose of deleting the fragment between the two sites. lox66 and lox71 are another pair of recognition sites for Cre recombinase. Under the action of Cre recombinase, they can recombine (the new site formed after recombination is lox72, which is also an inactive site, meaning it cannot be recognized by Cre recombinase to undergo recombination), thus achieving the purpose of deleting the fragment between the two sites. Furthermore, the loxF1 / loxF2 and lox66 / lox71 recognition sites are independent of each other and will not undergo cross-recombination. roxL1 and roxR1 are a pair of recognition sites for Dre recombinase. Under the action of Dre recombinase, they can recombine (the new site formed after recombination is inactive, meaning it cannot be recognized by Dre recombinase to undergo recombination), thus achieving the purpose of deleting the fragment between the two sites. The nox site is the recognition site of Nigri recombinase. Under the action of Nigri recombinase, plasmids carrying the nox site are integrated into the pre-knocked nox site on the host genome.

[0384] Table 3. Fragment Sequences

[0385] Example 3: Construction of Targeting Plasmid

[0386] 3-1) Construction of pKNF-MVA1

[0387] Using universal primers P1-up / P2-down and plasmid pKNF-1 as a template, the KNF-1 fragment was amplified by PCR, and the large fragment of the vector KNF-1 was recovered. This fragment can be stored for a long time at -80℃.

[0388] The artificially synthesized MVA1 gene fragment (approximately 5500 bp, sequence 5'-3' is P119(SEQ ID NO:24)-mvaE(GenBank accession number AF290092.1(1479..3890), 29-AUG-2000)-mvaS(GenBank accession number AF290092.1(142..1293)(-), 29-AUG-2000)-mvk(GenBank accession number NZ_CP075209.1(732139..733032), 13-JAN-2024)-idi(GenBank accession number Using NC_000913.3(3033065..3033613), 09-MAR-2022)-TrrnB), with MVA1-up and MVA1-down as primers and the MVA1 fragment as a template, the fragment P-MVA1 containing universal adapters P1 and P2 was amplified by high-fidelity TransStart FastPfu DNA polymerase (Beijing TransGen Biotech Co., Ltd., product catalog AP221). The target fragment P-MVA1 was recovered by agarose gel electrophoresis. The above P-MVA1 fragment was ligated to the KNF-1 fragment using the Gibson assembly method to obtain the ligation product. The ligation product was electrotransformed into E. coli BW25141 competent cells, clones were selected, and clones that could amplify the 1000bp target fragment were identified by primer P3 / MVA1-S and sequenced. The plasmid was extracted from the correctly identified positive clones and named pKNF-MVA1.

[0389] pKNF-MVA1 is a recombinant plasmid obtained by inserting the MVA1 gene fragment between the P1 and P2 sites of the pKNF-1 plasmid. Its structure from the 5' to the 3' end is: loxF1-nox-oriR6k-Kan-lox71-P3-P1-MVA1-P2.

[0390] 3-2) Construction of pSNL-MVA2

[0391] An artificially synthesized MVA2 gene fragment (approximately 3000 bp, sequence 5'-3' is P119-pmk(GenBank accession number NC_001145.3(712316..713671), 28-JUN-2024)-mvd(GenBank accession number NC_001146.8(701895..703085), 28-JUN-2024)-TrrnB) was used as primers for MVA2-up and MVA2-down. Using the MVA2 fragment as a template, the fragment P-MVA2 containing universal adapters P1 and P2 was amplified by high-fidelity TransStart FastPfu DNA polymerase (Beijing TransGen Biotech Co., Ltd., product catalog AP221). The target fragment P-MVA2 was recovered by agarose gel electrophoresis. The P-MVA2 fragment and the SNL-1 fragment (ablated by PCR amplification of plasmid pSNL-1 using universal primers P1-up / P2-down) were ligated using the Gibson assembly method to obtain the ligation product. The ligation product was electroporated into *E. coli* BW25141 competent cells. Clones were selected, and clones capable of amplifying the 1000 bp target fragment were identified using primers P3 / MVA2-S and sequenced. Positive clones were selected, and plasmids were extracted and named pSNL-MVA2.

[0392] pSNL-MVA2 is a recombinant plasmid obtained by inserting the MVA2 gene fragment between the P1 and P2 sites of the pSNL-1 plasmid. Its structure from the 5' to the 3' end is: lox66-nox-oriR6k-Str-loxF2-P3-P1-MVA2-P2.

[0393] The primers used are shown in Table 4.

[0394] Table 4. Primers and DNA fragments used in this implementation

[0395] Example 4: Integration of the MVA1 fragment into the chromosome of strain BN-01

[0396] 4-1) Preparation of Escherichia coli BN-01 (Example 1) electroporation competent cells. The pW-NC01 plasmid obtained in Example 2 was transformed into BN-01 cells by electroporation and cultured on ampicillin plates at 30°C; single colonies were picked and designated as strain BN01-W. The pW-NCD plasmid prepared in Example 2 was transformed into BN-01 cells by electroporation and cultured on ampicillin plates at 30°C; single colonies were picked and designated as strain BN01-D.

[0397] 4-2) Scheme 1: Using strain BN01-W, integrate the MVA1 fragment into the chromosome of strain BN-01.

[0398] 4-2a) [On the night of day 0] Inoculate the BN01-W strain into LB medium containing 50 mg / L ampicillin and incubate overnight.

[0399] 4-2b) [Day 1 Morning] Overnight cultured bacterial cells were inoculated into liquid LB medium (containing 50 mg / L ampicillin and 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells (2 hours). The plasmid pKNF-MVA1 obtained in Example 3 above was electrotransformed into BN01-W competent cells, cultured at 30°C for 1 hour, spread on solid LB culture dishes containing 50 mg / L ampicillin and 50 mg / L kanamycin, and incubated overnight at 30°C (16 hours).

[0400] 4-2c) [Morning of Day 2] Pick a single colony from the plate and incubate it at 30°C for 6 hours in LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin. Simultaneously with inoculation, place the picked single colony into a PCR reaction system containing primers lpxM-up-F284 (sequence shown in Table 1) / MVA1-S (sequence shown in Table 4) for colony PCR amplification. If the band size is approximately 3500 bp, the MVA-1 fragment has been successfully integrated at the lpxM site. That afternoon, transfer the positive clones inoculated in the morning to liquid LB medium containing 50 mg / L ampicillin. When OD600 = 0.2 (1 hour), add the inducer dehydrated tetracycline to a final concentration of 1 μM to induce the expression of the recombinase Cre. Continue incubation at 30°C for 3 hours, streak onto solid LB medium containing 50 mg / L ampicillin, and incubate at 30°C.

[0401] 4-2d) [Morning of Day 3] After single colonies have grown, pick a single colony and incubate it in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD600 = 1 (4 hours). Transfer it to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, and continue incubation at 30°C for 2 hours. During this period, perform PCR amplification on the liquid culture using the verification primers lpxM-up-F284 (sequence shown in Table 1) / lpxM-down-R195 (sequence shown in Table 1). If the PCR amplification product using lpxM-up-F284 / lpxM-down-R195 primers is approximately 7500 bp in size, and does not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it is a positive clone, designated as strain M1-W. In strain M1-W, the chromosome integrates one copy of the MVA-1 fragment, while still containing the pW-NC01 plasmid.

[0402] 4-3) Scheme 2: Using strain BN01-W, integrate the MVA1 fragment into the chromosome of strain BN-01, and optimize the process.

[0403] 4-3a) [On the night of day 0] Inoculate the BN01-W strain into LB medium containing 50 mg / L ampicillin and incubate overnight.

[0404] 4-3b) [Day 1 Morning] Overnight cultured bacterial cells were inoculated into LB medium (containing 50 mg / L ampicillin and a final concentration of 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells (2 hours). The plasmid pKNF-MVA1 obtained in Example 3 above was electrotransformed into BN01-W competent cells and cultured at 30°C for 2 hours in LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin. 100 μL of the culture was then transferred to 5 mL of LB medium containing 50 mg / L ampicillin and 1 μM tetracycline and cultured at 30°C for 2 hours. After centrifugation, the cells were spread onto solid LB medium containing 50 mg / L ampicillin and cultured overnight at 30°C.

[0405] 4-3c) [Morning of Day 2] Pick a single colony and incubate it in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD600 = 1 (4 hours). Then transfer it to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin and continue incubation at 30°C for 2 hours. During this period, perform PCR amplification on the liquid culture using the verification primers lpxM-up-F284 / lpxM-down-R195. If the PCR amplification product using lpxM-up-F284 / lpxM-down-R195 as primers is approximately 7500 bp in size, and does not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it is a positive clone, designated as strain M1-W. In strain M1-W, the chromosome integrates one copy of the MVA-1 fragment, and it still contains the pW-NC01 plasmid.

[0406] 4-4) Scheme 3: Using strain BN01-D, integrate the MVA1 fragment into the chromosome of strain BN-01.

[0407] 4-4a) [On the night of day 0], inoculate the BN01-D strain into LB medium containing 50 mg / L ampicillin and incubate overnight at 30°C.

[0408] 4-4b) [Day 1 Morning] Overnight cultured bacterial cells were inoculated into LB medium (containing 50 mg / L ampicillin and 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells (2 hours). The plasmid pKNF-MVA1 obtained in Example 3 above was electrotransformed into BN01-D competent cells. After culturing at 30°C for 1 hour, the cells were spread on solid LB culture dishes containing 50 mg / L ampicillin and 50 mg / L kanamycin and incubated overnight at 30°C (16 hours).

[0409] 4-4c) [Morning of Day 2] Pick a single colony from the plate and inoculate it into liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin. Incubate at 30°C for 6 hours. Simultaneously, place the picked single colony into a PCR reaction system containing primers lpxM-up-F284 (sequence shown in Table 1) / MVA1-S (sequence shown in Table 4) for colony PCR amplification. If the band size is approximately 3500 bp, the MVA-1 fragment has been successfully integrated at the lpxM site. That afternoon, transfer the positive clones inoculated in the morning into liquid LB medium containing 50 mg / L ampicillin. When OD600 = 0.2 (1 hour), add salicylic acid to a final concentration of 1 μM to induce the expression of the recombinase Cre. Continue incubation at 30°C for 3 hours, then plate onto solid LB medium containing 50 mg / L ampicillin and incubate at 30°C.

[0410] 4-4d) [Morning of Day 3] After single colonies have grown, pick them and incubate them separately in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD600 = 1 (4 hours). Then transfer them to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, and continue incubation at 30°C for 2 hours. During this period, perform PCR amplification on the liquid culture using the verification primers lpxM-up-F284 / lpxM-down-R195. If the PCR amplification product using lpxM-up-F284 / lpxM-down-R195 is approximately 7500 bp in size and does not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it is a positive clone, designated as strain M1-D. Strain M1-D contains one copy of the MVA-1 fragment integrated into the chromosome and also retains the pW-NCD plasmid.

[0411] 4-5) Scheme 4: Using strain BN01-D, integrate the MVA1 fragment into the chromosome of strain BN-01, process 2.

[0412] 4-5a) [On the night of day 0], inoculate the BN01-D strain into LB medium containing 50 mg / L ampicillin and incubate overnight at 30°C.

[0413] 4-5b) [Day 1 Morning] Overnight cultured bacterial cells were inoculated into LB medium (containing 50 mg / L ampicillin and 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells (2 hours). The plasmid pKNF-MVA1 obtained in Example 3 above was electrotransformed into BN01-D competent cells. After culturing at 30°C for 1 hour, the cells were spread on solid LB culture dishes containing 50 mg / L ampicillin and 50 mg / L kanamycin and incubated overnight at 30°C (16 hours).

[0414] 4-5c) [Morning of Day 2] Pick a single colony from the plate and inoculate it into liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin. Incubate at 30°C for 6 hours. Simultaneously, place the picked single colony into a PCR reaction system containing primers lpxM-up-F284 (sequence shown in Table 1) / MVA1-S (sequence shown in Table 4) for colony PCR amplification. If the band size is approximately 3500 bp, the MVA-1 fragment has been successfully integrated at the lpxM site. That afternoon, transfer the positive clones inoculated in the morning into liquid LB medium containing 50 mg / L ampicillin. When OD600 = 0.2 (1 hour), add salicylic acid to a final concentration of 1 μM to induce the expression of the recombinase Cre. Continue incubation at 30°C for 20 hours.

[0415] 4-5d) [Afternoon of Day 3] Spread the culture onto solid LB medium containing 50 mg / L ampicillin and incubate overnight at 30°C.

[0416] 4-5e) [Morning of Day 4] After single colonies have grown, pick them and incubate them separately in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD600 = 1 (4 hours). Then transfer them to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, and continue incubation at 30°C for 2 hours. During this period, perform PCR amplification on the liquid culture using the verification primers lpxM-up-F284 / lpxM-down-R195. If the PCR amplification product using lpxM-up-F284 / lpxM-down-R195 is approximately 7500 bp in size, and does not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it is a positive clone, designated as strain M1-D. Strain M1-D contains one copy of the MVA-1 fragment integrated into the chromosome, and also retains the pW-NCD plasmid.

[0417] Results 1. Positive rates of steps 4-2c), 4-4c), and 4-5c) (i.e., integration of the MVA-1 fragment at the lpxM site was completed, but the resistance was not eliminated).

[0418] In the above steps, the positive clones identified after selection were calculated as follows: 15 clones were randomly selected, and the positive rate = number of positive clones / total number of clones selected. Cfu / μg DNA was defined as the total number of clones grown after transforming 1 microgram of the target plasmid fragment. The experiment was repeated three times. The results are shown in Table 5:

[0419] Table 5

[0420] Result 2. Positive rates of steps 4-2d), 4-3c), 4-4d), and 4-5e) (i.e., chromosome integration with one copy of the MVA-1 fragment and elimination of resistance, but plasmid still present).

[0421] In the above steps, the number of clones identified as positive after selection was calculated as follows: 15 clones were randomly selected, and the positive rate was calculated as: positive rate = number of positive clones / total number of clones selected. The experiment was repeated three times. The results are shown in Table 6.

[0422] Table 6

[0423] Result 3. Cycle of Schemes 1-4

[0424] See Table 7.

[0425] Table 7

[0426] Based on results 1, 2, and 3, the gene integration schemes based on pW-NC01 and pW-NCD plasmids provided in this invention can achieve highly efficient integration of exogenous fragments. Among them, after optimization, pW-NC01 can enable efficient expression of Cre enzyme, which can shorten the gene integration-elimination of selection marker cycle to 2 days (i.e., scheme 2).

[0427] Example 5: Using strain BN01-W, MVA1 and MVA2 fragments were sequentially and alternately integrated into the chromosome of strain BN01.

[0428] 5-1) [On the night of day 0] Inoculate the BN01-W strain into LB medium containing 50 mg / L ampicillin and incubate overnight at 30°C.

[0429] 5-2) [Day 1 Morning] Overnight cultured bacterial cells were inoculated into LB medium (containing 50 mg / L ampicillin and a final concentration of 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells. The plasmid pKNF-MVA1 obtained in Example 3 above was electrotransformed into BN01-W competent cells and cultured at 30°C for 2 hours in LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin. Then, 100 μL of the culture medium was transferred to 5 mL of LB medium containing 50 mg / L ampicillin and 1 μM tetracycline and cultured at 30°C for 2 hours. After centrifugation, the cells were spread onto solid LB medium containing 50 mg / L ampicillin and cultured overnight at 30°C.

[0430] 5-3) [Morning of Day 2] Single colonies were picked and cultured in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD600 = 1. The liquid cultures were then amplified by PCR using Lamp Maxter Mix enzyme (Novozymes, catalog number: P312-02) with primers lpxM-up-F / lpxM-down-R (sequences shown in Table 1). Simultaneously, the amplified products were transferred to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin and cultured at 30°C for 2 hours. If the PCR product was 5500 bp in size and did not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it was considered a positive clone. Further sequencing was performed to confirm the PCR product. The positive clone was designated strain M1-W. Strain M1-W contained one copy of the MVA-1 fragment integrated into the chromosome and also retained the pW-NC01 plasmid. Strains M1-W were inoculated into LB medium containing 50 mg / L ampicillin and cultured overnight at 30°C.

[0431] 5-4) [Morning of Day 3] The M1-W cells cultured overnight were inoculated into LB medium (containing 50 mg / L ampicillin and a final concentration of 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells. The plasmid pSNL-MVA2 obtained in Example 3 was electrotransformed into the M1-W competent cells and cultured at 30°C for 2 hours in LB medium containing 50 mg / L ampicillin and 50 mg / L streptomycin. Then, 100 μL of the culture medium was transferred to 5 mL of LB medium containing 50 mg / L ampicillin and 1 μM tetracycline and cultured at 30°C for 2 hours. After centrifugation, the cells were spread onto solid LB medium containing 50 mg / L ampicillin and cultured overnight at 30°C.

[0432] 5-5) [Day 4 morning] Pick single colonies and incubate them in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD.600 =1. Using lpxM-up-F / lpxM-down-R (sequences shown in Table 1) as primers, PCR amplification was performed on the liquid culture using Lamp Maxter Mix enzyme. Simultaneously, the culture was transferred to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L streptomycin and incubated at 30°C for 2 hours. If the PCR amplification product was 8500 bp in size and did not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it was considered a positive clone. Further sequencing was performed to confirm the PCR product, and the positive clone was designated as strain M2-W. Strain M2-W contained one copy of the MVA-1 fragment and one copy of the MVA-2 fragment integrated into its chromosome, and also retained the pW-NC01 plasmid. M2-W was inoculated into LB medium containing 50 mg / L ampicillin and incubated overnight at 30°C.

[0433] 5-6) [Morning of Day 5] Overnight cultured bacterial cells were inoculated into LB medium (containing 50 mg / L ampicillin and a final concentration of 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells. The plasmid pKNF-MVA1 obtained in Example 3 was electrotransformed into M2-W competent cells and cultured at 30°C for 2 hours in LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin. Then, 100 μL of the culture was transferred to 5 mL of LB medium containing 50 mg / L ampicillin and 1 μM tetracycline and cultured at 30°C for 2 hours. After centrifugation, the cells were spread onto solid LB medium containing 50 mg / L ampicillin and cultured overnight at 30°C.

[0434] 5-7) [Morning of Day 6] Pick single colonies and incubate them separately in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD. 600 =1. Using lpxM-up-F / lpxM-down-R (sequences shown in Table 1) as primers, PCR amplification was performed on the liquid culture using Lamp Maxter Mix enzyme. Simultaneously, the culture was transferred to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L streptomycin and incubated at 30°C for 2 hours. If the PCR amplification product was 14000 bp in size and did not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it was considered a positive clone. Further sequencing was performed on the PCR product to confirm its identity. The positive clone was designated as strain M3-W. Strain M3-W contained two copies of the MVA-1 fragment and one copy of the MVA-2 fragment integrated into its chromosome, and also retained the pW-NC01 plasmid. M3-W was inoculated into LB medium containing 50 mg / L ampicillin and incubated overnight at 30°C.

[0435] 5-8) [Morning of Day 7] Overnight cultured bacterial cells were inoculated into LB medium (containing 50 mg / L ampicillin and a final concentration of 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells. The plasmid pSNL-MVA2 obtained in Example 3 was electrotransformed into M3-W competent cells and cultured at 30°C for 2 hours in LB medium containing 50 mg / L ampicillin and 50 mg / L streptomycin. Then, 100 μL of the culture was transferred to 5 mL of LB medium containing 50 mg / L ampicillin and 1 μM tetracycline and cultured at 30°C for 2 hours. After centrifugation, the cells were spread onto solid LB medium containing 50 mg / L ampicillin and cultured overnight at 30°C.

[0436] 5-9) [Morning of Day 8] Pick single colonies and incubate them separately in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD. 600 =1. Using lpxM-up-F / lpxM-down-R (sequences shown in Table 1) as primers, PCR amplification was performed on the liquid culture using Lamp Maxter Mix enzyme. Simultaneously, the culture was transferred to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L streptomycin and incubated at 30°C for 2 hours. If the PCR amplification product was 17000 bp in size and did not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it was considered a positive clone. Further sequencing was performed on the PCR product to confirm its identity. The positive clone was designated strain M4-W. Strain M4-W contained two copies of the MVA-1 fragment and two copies of the MVA-2 fragment integrated into its chromosome, and also retained the pW-NC01 plasmid.

[0437] 5-10) Single colonies of M1-W, M2-W, M3-W, and M4-W were picked and incubated overnight at 42°C. Then, they were streaked onto antibiotic-free solid LB medium and incubated at 37°C for 12 hours. After single colonies grew, they were picked and inoculated onto antibiotic-free and solid LB medium containing 50 mg / L ampicillin, respectively, and incubated overnight at 37°C. If no growth was observed on solid LB medium containing 50 mg / L ampicillin, then the pW-NC01 plasmid was successfully eliminated, and the obtained single colonies were strains M1, M2, M3, and M4, respectively.

[0438] Results 4. Positive rates in steps 5-3), 5-5), 5-7), and 5-9).

[0439] In the above steps, the number of clones identified as positive after selection was calculated as follows: 15 clones were randomly selected, and the positive rate = number of positive clones / total number of clones selected. Cfu / μg DNA was defined as the total number of clones grown after transforming 1 microgram of the target plasmid fragment. The experiment was repeated three times. The results are shown in Table 8:

[0440] Table 8

[0441] The results above show that this method can achieve four consecutive chromosome integrations with high efficiency within 8 days.

[0442] Example 6: Large Fragment Integration

[0443] In Example 2, the targeting plasmid employed a replication initiation site based on the R6k sequence. This plasmid requires construction and preparation in a host expressing the pir protein, such as BW25141. However, due to the low copy number, the efficiency of plasmid construction and preparation significantly decreases when the target plasmid size exceeds 10kb. Therefore, the plasmid was modified to construct the pKNF-2 / pSNL-2 plasmid system. This plasmid system's replication initiation site contains two parts: a ColE replication initiation site (including the tetA sequence, which facilitates negative selection during sequence elimination) and a high-copy-rate repA1 replication protein binding site. During construction, the ColE replication initiation site ensures both sufficient copy number and construction efficiency for large-fragment plasmid construction, allowing for successful construction in common cloning hosts such as DH5α and Top10 E. coli, as well as various other hosts. In strain BCR-01, the ColE replication origin site can be eliminated using the Cre enzyme, resulting in a repA1-dependent conditional replication plasmid, thus eliminating false positives from autonomously replicating plasmids during the targeting process. Simultaneously, the repA1-dependent replication origin site (oriSC101) significantly increases the plasmid copy number, facilitating the preparation of higher concentrations of targeting plasmids and simplifying the preparation of large-fragment plasmids.

[0444] 6-1) Construction of strain BCR-01

[0445] Referring to the procedure in Example 1, the Cre recombinase gene with an arabinose promoter and the gene encoding the repA1 replication initiation protein with a trc promoter were integrated into the position of the myristoyltransferase gene lpxM in the chromosome of Escherichia coli BW25113 strain, and the lpxM gene was knocked out. The specific operation is as follows:

[0446] The artificially synthesized gene CR-01 targeting fragment, with the sequence lpxMup1000-ParaBAD-Cre-TrrnB-Ptrc-repA1-TrrnB-lpxMdown1000 (each part is connected end to end in sequence, and the specific sequences of each part are shown in Table 3 or Table 10 respectively), was used to amplify and enrich the CR-01 targeting fragment by PCR using primers CR-01F / CR-01R (sequences are shown in Table 10).

[0447] The target fragment CR-01 obtained above and the plasmid pTarget-lpxM obtained in steps 1-3) of Example 1 were co-transformed into the electrocompetent cells obtained in steps 1-5) of Example 1. After culturing at 30°C for 1 hour, the cells were spread on solid LB culture dishes containing 50 mg / L kanamycin and 50 mg / L streptomycin and placed in an incubator at 30°C until single clones grew.

[0448] The obtained single colonies were verified by colony PCR. The verification primers were lpxM-up-F284 / lpxM-down-R195 (sequences are shown in Table 1). If the band size was only about 5000 bp, it means that integration at the lpxM site was completed. The correct single colony was selected and the plasmid was removed according to the steps 1-8) to 1-9) in Example 1. The obtained single colony is the BCR-01 strain.

[0449] The BCR-01 strain is obtained by replacing the lpxM gene in the genome of Escherichia coli BW25113 with the fragment “ParaBAD-Cre-TrrnB-Ptrc-repA1-TrrnB”, while keeping other sequences unchanged.

[0450] 6-2) Construction of large fragment construction tool plasmids

[0451] The tool plasmids used can be synthesized from the whole genome or spliced ​​together. Relevant information is shown in Tables 9 and 10.

[0452] Table 9. Plasmid Information Note: loxF1 and loxF are a pair of recognition sites for Cre recombinase. They can recombine under the action of Cre recombinase (leaving the loxF1 site after recombination), thus achieving the purpose of deleting the fragment between the two sites; lox66 and loxP are another pair of recognition sites for Cre recombinase. They can recombine under the action of Cre recombinase (leaving the lox66 site after recombination), thus achieving the purpose of deleting the fragment between the two sites.

[0453] Table 10. Fragment and Primer Information

[0454] 6-2a) Construction of large fragment targeting plasmids

[0455] Using universal primers P1-up / P2-down (sequences shown in Table 4), the KNF-2 fragment was amplified by PCR using plasmid pKNF-2 as a template. The large KNF-2 fragment was recovered and can be stored at -80℃ for a long time. Using AV1-up and AV1-down primers (sequences shown in Table 11), the fragment P-AV1 containing universal adapters P1 and P2 was amplified by PCR using high-fidelity TransStart FastPfu DNA polymerase (Beijing TransGen Biotech Co., Ltd., product catalog AP221) as a template. The target fragment P-AV1 (sequence P1-avermectin gene cluster (AB032367.1 12,020-64,957(+)1999.9.18)-P2) was recovered by agarose gel electrophoresis. The P-AV1 fragment was ligated to the KNF-1 fragment using the Gibson assembly method to obtain the ligation product. Prepare BCR-01 strain competent cells obtained in 6-1) by electroporation. Electroporate the above ligation product into BCR-01 competent cells, spread them on a plate containing 50 mg / L kanamycin, and incubate them in a 37°C incubator until single clones grow. Select single clones for culture, and use primers P3 / AV1-S (sequences are shown in Tables 4 and 11) to identify clones that can amplify the 1000 bp target fragment as positive clones.

[0456] 6-2b) Elimination of ColE replication origin sites

[0457] The BCR-01 strain containing the positive plasmid obtained above was transferred to LB medium containing 50 mg / L kanamycin and grown for 2 hours. After adding 0.2% arabinose to a final concentration, the culture was continued for another 2 hours. The culture was then plated on LB medium containing 50 mg / L kanamycin and 50 μM nickel chloride and incubated overnight at 37°C. Single clones were picked and transferred to LB medium containing 50 mg / L kanamycin and ITPG and cultured at 37°C. PCR identification was performed using primers ColE1-F / CoE1-R (sequences shown in Table 11). If the target band could not be amplified, it indicated that the ColE1 replication origin site had been eliminated, and the positive plasmid was named pKNFS2-AV1. pKNFS2-AV1 was extracted as the targeting plasmid. pKNFS2-AV1 inserts a gene cluster containing a 50kb fragment, with the following structure from the 5' to the 3' end: loxF1-nox-oriSC101-Kan-lox71-P3-P1-AV1-P2.

[0458] Using the same method, pKNFS2-AV2, pKNFS2-AV3, pKNFS2-AV4, and pKNFS2-MVA1 were constructed respectively. The plasmids described above contain fragments of AV2 (AV2-down was used instead of AV1-down during construction, sequence: Genbank AB032367.1 12,020-37,320(+)1999.9.18), AV3 (AV3-down was used instead of AV1-down during construction, sequence: Genbank AB032367.1 12,020-27,240(+)1999.9.18), AV4 (AV4-down was used instead of AV1-down during construction, sequence: Genbank AB032367.1 12,020-22,020(+)1999.9.18), and MVA1 (MVA1-up / MVA1-down / MVA1-s were used instead of AV1-up / AV1-down / AV1-S during construction, primer sequences are shown in Table 4, and the MVA1 sequence is shown in Example 3).

[0459] The relevant primer sequence information is shown in Table 11.

[0460] Table 11. Primer Information

[0461] 6-2c) AV1 large fragment integrated into chromosome.

[0462] The method follows step 4-2) of Example 4, integrating the above-mentioned exogenous fragment into the chromosome of BN-01. The positive clone rate is then calculated.

[0463] The primers AV-F / AV-R shown in Table 11 were used to identify the PCR products. A PCR amplification product size of approximately 4000 bp was considered positive.

[0464] Results 5. Comparison of plasmid construction efficiency and plasmid preparation concentration between pKNFS2-MVA1 and pKNF-MVA1.

[0465] pKNF-MVA1 and pKNFS2-MVA1 were constructed according to the procedures described in steps 3-1) and 6-2b) above, respectively. The number of clones after electroconversion is shown in Table 12.

[0466] Table 12

[0467] The above results indicate that using the pKNFS2 plasmid can improve plasmid construction efficiency. Furthermore, this method produces plasmids with high concentrations.

[0468] Results 6. Targeting efficiency of pKNFS2-AV2, pKNFS2-AV2, pKNFS2-AV3, pKNFS2-AV4, and pKNFS2-MVA1 (i.e., a result analysis of section 6-2c).

[0469] The positive clone rates of different fragments were compared. The results are shown in Table 13: The results show that the number of transformed clones decreases as the size of the exogenous fragment increases. This method can integrate exogenous fragments up to 50kb.

[0470] Table 13

[0471] Example 7: CRISPR-mediated integration

[0472] The tool plasmids used can be synthesized from the whole genome or spliced ​​together. Relevant information is shown in Tables 2 and 3.

[0473] 7-1) Prepare Escherichia coli BW25113 electroporation competent cells, and transfer the pW-NC02 plasmid (Table 14) into them by electroporation. Culture them on ampicillin plates at 30°C. Select single clones and record them as strain BW25113-CW.

[0474] Table 14. Plasmid Information

[0475] 7-2) CRISPR-mediated sequential integration.

[0476] 7-2a) [On the night of day 0] Inoculate the BW25113-CW strain into LB medium containing 50 mg / L ampicillin and incubate overnight.

[0477] 7-2b) [Day 1 Morning] Overnight cultured bacterial cells were inoculated into LB medium (containing 50 mg / L ampicillin and 1 μM salicylic acid) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells. The targeting fragment L3 (SEQ ID NO:26) and plasmid pTarget-lpxM prepared in 1-2) were electrotransformed into BW25113-CW competent cells and incubated overnight at 30°C on LB medium plates containing 50 mg / L ampicillin and 50 mg / L streptomycin.

[0478] 7-2c) [Morning of Day 2] Pick single colonies from overnight plates and inoculate them into LB medium (containing 50 mg / L ampicillin, final concentration 0.5 mM IPTG), and incubate at 30°C for 3 hours. Simultaneously, perform colony PCR amplification. Verify the obtained single colonies using colony PCR with the primers lpxM-up-F284 / lpxM-down-R195 (sequences shown in Table 1). If the band size is only about 2000 bp, it indicates successful integration of nox-lox66 at the lpxM site. Transfer the corresponding liquid culture to LB medium (containing 50 mg / L ampicillin, final concentration 0.2% L-arabinose), and incubate at 30°C for 3 hours to prepare electrotransformation competent cells. The plasmid pKNF-MVA1 obtained in Example 3 was electroporated into competent cells. After culturing at 30°C for 2 hours in LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, 100 μL of the culture medium was transferred to 5 mL of LB medium containing 50 mg / L ampicillin and 1 μM tetracycline. After culturing at 30°C for 2 hours, the culture was centrifuged and spread onto solid LB medium containing 50 mg / L ampicillin. The cells were then incubated overnight at 30°C.

[0479] 7-3d) [Morning of Day 3] Pick single colonies and incubate them in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD. 600 =1, transferred to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, and incubated at 30°C for 2 hours. Simultaneously, PCR amplification was performed on the liquid culture using the verification primers lpxM-up-F284 / lpxM-down-R195. If the PCR amplification product using lpxM-up-F284 / lpxM-down-R195 (sequence shown in Table 1) was approximately 4000 bp in size, and did not grow on liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, it was a positive clone, designated as strain M1-W. Strain M1-W contained one copy of the MVA-1 fragment integrated into the chromosome, and also retained the pW-NC02 plasmid.

[0480] Result 7. Detection of positive clones in 7-3d)

[0481] In 7-3d), the number of positive clones was 100%. This demonstrates that the method can effectively achieve exogenous gene integration within 3 days. If different targeting fragments and pTarget plasmids are used in 7-3b), rapid gene integration at any site can be achieved.

[0482] The fragment sequences involved in Examples 8-15 below are shown in Table 15. The plasmid information involved in the following examples is shown in Table 16. The primer sequences involved in the following examples are shown in Table 17.

[0483] Table 15. Fragment sequences involved in embodiments 8-15 of the present invention

[0484] Table 16. Plasmid information involved in Examples 8-15 of the present invention

[0485] Table 17. Primer sequences involved in Examples 8-15 of the present invention

[0486] Example 8: Construction of pUCK01 plasmid

[0487] 8-a) Preparation of pU and CK01 fragments

[0488] The plasmid backbone pU fragment and the chloramphenicol-kanamycin resistance gene fragment CK01 were synthesized by Qingke Biotechnology. The 5'-3' sequence of the pU fragment is the ColE replication origin site (SEQ ID NO:138). Using this fragment as a template, PCR amplification was performed using primers pU-F / pU-R to prepare the pU fragment. The 5'-3' sequence of the CK01 fragment is the kanamycin resistance gene (SEQ ID NO:123) and the chloramphenicol resistance gene (SEQ ID NO:125) linked together. Using this fragment as a template, PCR amplification was performed using primers CK01-F / CK01-R to prepare the CK01 fragment, wherein primer CK01-R contains the lox71 (SEQ ID NO:111) site.

[0489] All the above PCRs were performed using high-fidelity TransStart FastPfu DNA polymerase (Beijing TransGen Biotech Co., Ltd., product catalog AP221), and the target fragments were recovered by agarose gel electrophoresis.

[0490] 8-b) The CK01 fragment and pU fragment were ligated using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods. 2009; 6(5):343-345) to obtain the recombinant expression vector pUCK01. Escherichia coli DH5α competent cells (purchased from Beijing TransGen Biotech Co., Ltd., product catalog: CD201) were transformed using the CaCl2 method. The cells were evenly spread on LB plates containing kanamycin and chloramphenicol and incubated overnight at 37°C. Clones were selected, and those capable of amplifying the target fragment were identified using primers pU-3 / CK01-R and sequenced. Positive clones were selected, and plasmids were extracted to obtain the positive plasmid, named pUCK01. The pUCK01 plasmid sequentially contains the kanamycin resistance gene, chloramphenicol resistance gene, lox71 site, and ColE replication origin site.

[0491] Example 9: Construction of a pUCK-N library containing the nox mutation site

[0492] 9-a) Design of the nox site mutation library. The nox site consists of a 34-base sequence (SEQ ID NO: 97), comprising upstream and downstream flanking regions composed of 13 symmetrical bases on both sides and a core region composed of 8 asymmetrical bases in the middle. We randomly mutated the 8 bases in the core region to form the sequence noxM (SEQ ID NO: 137).

[0493] 9-b) Preparation of pUC and KN fragment libraries

[0494] Using the pUCK01 plasmid constructed in Example 8 as a template, PCR amplification was performed using primers pUC-F / pU-R to prepare the pUC fragment. The 5'-3' sequence of the pUC fragment is: chloramphenicol resistance gene, lox71 site, and ColE replication origin site.

[0495] Using the pUCK01 plasmid constructed in Example 8 as a template, PCR amplification was performed using primers K-NF / K-NR to prepare a KN fragment library. The noxM sequence was introduced into primer K-NF, and lox66 and wild-type nox sequences were introduced into K-NR. The DNA sequence of the KN fragment library (5'-3') is as follows: noxM, kanamycin resistance gene, lox66, nox.

[0496] All the above PCRs were performed using high-fidelity TransStart FastPfu DNA polymerase (Beijing TransGen Biotech Co., Ltd., product catalog AP221), and the target fragments were recovered by agarose gel electrophoresis.

[0497] 9-c) The above KN fragment library was ligated with the pUC fragment using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods. 2009; 6(5):343-345) to obtain the recombinant expression vector library pUCK-N. Escherichia coli DH5α competent cells (purchased from Beijing TransGen Biotech Co., Ltd., product catalog: CD201) were transformed using the CaCl2 method. The cells were evenly spread on LB agar plates containing chloramphenicol and kanamycin and incubated overnight at 37°C. The clones on the plates were washed with physiological saline and then added to liquid LB medium containing chloramphenicol and kanamycin, and incubated overnight at 37°C. The plasmids were extracted, and the resulting mixed plasmid was the pUCK-N library. The sequence of the pUCK-N library plasmid, from 5' to 3', consists of: noxM, kanamycin resistance gene, lox66, nox, chloramphenicol resistance gene, lox71, and ColE replication origin site.

[0498] Example 10: Screening of Nox mutants with orthogonal functions

[0499] Preparation of 10-a)pW-NC01 plasmid

[0500] The pW-NC01 plasmid was synthesized using the whole genome (Table 16).

[0501] 10-b) First round of screening.

[0502] pUCK-N was transformed into Escherichia coli BW25113 containing the pW-NC01 plasmid to obtain a strain library.

[0503] In the first round, the library was transferred to LB medium containing 50 mg / L ampicillin and 17 mg / L chloramphenicol, 50 mg / L ampicillin and 0.2% arabinose and cultured at 30°C for 16 hours. Then it was transferred to the same medium and cultured at 30°C for 10 hours. Finally, it was transferred to LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin and cultured at 37°C for 16 hours to obtain the NOX-1 strain library that completed the first round of screening.

[0504] When the two Nox sites are located on the *E. coli* chromosome and plasmid respectively (in trans), homologous recombination occurs efficiently, integrating the entire plasmid fragment into the chromosome. When both Nox sites are located on the plasmid (in cis), homologous recombination occurs at a relatively low efficiency; extending the cell culture time can improve the recombination efficiency of in cis. However, if the Nox mutant cannot recombine with wild-type Nox, recombination will not occur even with extended cell culture time, and the cells can grow in kanamycin. Conversely, if the Nox mutant can react with wild-type Nox, it will not grow due to the elimination of kanamycin resistance. Therefore, even after prolonged cell culture, the Nox mutant in cells on kanamycin-containing medium cannot recombine with wild-type Nox. Thus, the Nox mutant sequences in the obtained NOX-1 strain library that completed the first round of screening all possess the above-mentioned functions.

[0505] 10-c) Second round of screening.

[0506] The NOX-1 library strain was transferred to LB medium containing 50 mg / L kanamycin, 50 mg / L ampicillin, and 0.2% arabinose and cultured at 30°C for 16 hours. Recombination between lox66 and lox71 on the NOX-1 library plasmid was induced using Cre recombinase, thereby eliminating the wild-type nox site and chloramphenicol resistance gene. The strain was then transferred again to LB medium containing 50 mg / L kanamycin, 50 mg / L ampicillin, and 0.2% arabinose and cultured at 30°C for 16 hours. The resulting plasmids were then plated onto LB medium containing 50 mg / L kanamycin, 50 mg / L ampicillin, and 0.2% arabinose until single clones emerged. The plasmids in the single clones of this strain contained only one nox mutant. The nox sequences of the two plasmid molecules may undergo homologous recombination with low efficiency under the action of Nigri recombinase. If the above situation occurs, because the plasmid has two or more replication origin sites, the replication efficiency of this type of plasmid is usually lower than that of normal plasmids, which leads to plasmid instability and affects bacterial growth, resulting in smaller colonies on plates. Therefore, selecting smaller colonies increases the probability of screening for nox mutants that still possess homologous recombination capabilities. The clones with the smaller colonies were transferred to LB medium containing 50 mg / L kanamycin. Sequencing was performed to obtain several nox mutants (SEQ ID NO:98 to SEQ ID NO:108).

[0507] Example 11: Construction of strains BN-1, BN-2, BN-3, BN-4…BN-12

[0508] 11-a) Construction of strain BN-1

[0509] The CRISPR-Cas9 method follows the procedure described in the literature "Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514." The integration recognition site "lox66-nox" is integrated into the myristoyltransferase gene lpxM in the chromosome of *E. coli* strain BW25113 (lox66 first, nox second in 5'-3' order), and the lpxM gene is then knocked out. The specific procedures are as follows:

[0510] 11-aI) Preparation of target fragment L01

[0511] Gene-synthesized DNA fragment (GenScript): Targeted fragment L01 (sequence SEQ ID NO: 129). Using lpxM-1 / lpxM-2 as primers and the gene-synthesized DNA fragment as a template, PCR amplification was performed to obtain the targeted fragment L01.

[0512] 11-a-II) Primers lpxM-N20-F / lpxM-N20-R were designed, and plasmid pTargetF (referring to the literature "Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514.") was used as a template for PCR amplification. The PCR product was treated with DpnI enzyme and then transformed into DH5α to obtain plasmid pTarget-lpxM.

[0513] The structure of plasmid pTarget-lpxM is achieved by replacing the sequence from position 227 to 246 of plasmid pTargetF with sequence 5'-ATGATCATACGATTGCGCGG-3' (the coding sequence of the sgRNA targeting the lpxM gene, SEQ ID NO:128).

[0514] 11-a-III) Prepare Escherichia coli BW25113 electrotransformed competent cells, and transfer pCAS plasmid (Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol. 2015; 81(7):2506-14.) into the cells by electrotransformation. The cells were cultured on kanamycin plates at 30°C. Single clones were picked and labeled as BW25113 / Pcas strain.

[0515] 11-a-IV) The obtained BW25113 / Pcas strain was inoculated into LB medium containing 50 mg / L kanamycin, and then 0.2% L-arabinose was added to the medium. The culture was carried out at 30°C for 3-4 hours to prepare electrotransformation competent cells.

[0516] 11-aV) The target fragment L01 and plasmid pTarget-lpxM obtained above were co-transformed into the electrocompetent cells obtained in step 11-a-IV). After culturing at 30°C for 1 hour, the cells were spread on solid LB culture dishes containing 50 mg / L kanamycin and 50 mg / L streptomycin and placed in an incubator at 30°C until single clones grew.

[0517] 11-a-VI) The single colonies obtained above were verified by colony PCR. The verification primers were lpxM-1 / lpxM-2. If the band size was only about 1000 bp, it means that lox66-nox was successfully integrated at the lpxM site. The correct single colonies were picked and cultured overnight at 30°C in liquid LB medium containing 50 mg / L kanamycin and 0.5 mM IPTG.

[0518] 11-a-VII) The overnight cultured bacterial suspension was streaked onto solid LB medium containing 50 mg / L kanamycin and incubated at 30°C. After single colonies grew, single colonies were picked and placed on solid LB medium containing 50 mg / L kanamycin and 50 mg / L kanamycin and streptomycin, respectively, and then incubated at 30°C. If the inoculated single colonies grew on solid LB medium containing 50 mg / L kanamycin but not on solid LB medium containing 50 mg / L streptomycin, then the pTarget-lpxM plasmid elimination was completed (the principle and process are described in the literature "Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514.").

[0519] (11-a-VIII) Single colonies of the pTarget-lpxM plasmid eliminated were picked and incubated overnight at 42°C on liquid LB medium. Then, they were streaked onto antibiotic-free solid LB medium and incubated at 37°C for 12 hours. After single colonies grew, they were picked and inoculated onto antibiotic-free and solid LB medium containing 50 mg / L kanamycin, respectively, and incubated overnight at 37°C. If no growth was observed on solid LB medium containing 50 mg / L kanamycin, then the pCAS plasmid elimination was successful (the principle and process are described in the reference "Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S: Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl Environ Microbiol 2015, 81:2506-2514."). The obtained single colonies are strain BN-1.

[0520] The BN-1 strain is obtained by replacing the lpxM gene in the genome of Escherichia coli BW25113 with the integration recognition site "lox66-nox" while keeping other sequences unchanged.

[0521] 11-b) Following the construction process of strain BN-1 in 11-a), strains BN-2, BN-3, BN-4…BN-n were constructed starting from strain BW25113. The difference was that throughout the entire process, the nox sequence was replaced with a nox mutant sequence (referred to as nox1, nox2…nox11 in sequence). The strains BN-2, BN-3, BN-4…BN-12 were obtained by replacing the lpxM gene in the genome of Escherichia coli BW25113 with the integration recognition sites “lox66-nox1”, “lox66-nox2”, “lox66-nox3”…“lox66-nox11” (in this operation, the lox66-nox sequence at positions 501-568 of SEQ ID NO:129 was replaced accordingly), while keeping other sequences unchanged.

[0522] Example 12: Construction of tool plasmids and plasmids pKNF-1, pKNF-2, pKNF-3, pKNF-4, ..., pKNF-11, pSNF-9, pCNF-11, pPNF-4, pSNL-1, pCNL-9, and pKNL-11

[0523] All plasmids were prepared using whole-gene synthesis or splicing methods, and relevant information is shown in Table 16. All genetic operations on the plasmids were performed in *E. coli* BW25141, and the plasmids were extracted and used for later use.

[0524] Example 13: Verification of the function of the nox mutant

[0525] 13-a) Preparation of Escherichia coli BN-1D, BN-2D, ... BN-12D electrotransformation competent cells.

[0526] Taking BN-1D as an example, the pW-NC01 plasmid was transformed into the BN-1 strain obtained in Example 11 by electroporation and cultured on ampicillin plates at 30°C. Single clones were picked and designated as strain BN1-1D. The BN-1D strain was cultured overnight in liquid LB medium, then inoculated into LB medium (containing 50 mg / L ampicillin and a final concentration of 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electroporated competent cells, thus obtaining BN-1D competent cells.

[0527] Replace strain BN-1 with strains BN-2, BN-3, ... BN-12 and obtain BN-1D, BN-2D, ... BN-12D electrocompetent cells respectively by following the exact same process.

[0528] 13-b) The plasmid pKNF-1 was transferred into competent cells BN-1D.

[0529] Plasmid pKNF-1 was electroporated into BN-1D competent cells. After culturing at 30°C for 1 hour, the cells were spread onto solid LB culture dishes containing 50 mg / L ampicillin and 50 mg / L kanamycin and incubated overnight at 30°C until single colonies emerged from the long plates.

[0530] 13-c) Identification of chromosome integrated with plasmid pKNF-1.

[0531] Single colonies were picked from the plate and incubated at 30°C for 6 hours in LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin. The bacterial culture was then placed in a PCR reaction system containing lpxM-up / Kan-R primers for colony PCR amplification. If the band size was approximately 1800 bp, integration of the pKNF-1 plasmid at the lpxM site was complete, and this was recorded as "YES". If integration failed, it was recorded as "NO".

[0532] 13-d) Following the procedures in 13-b) and 13-c), the different plasmids (pKNF-1, pKNF-2, pKNF-3, ... pKNF-11) were transferred into different competent cells (BN-1D, BN-2D, BN-3D, ... BN-12D), and the integration efficiency was recorded ("YES" or "NO").

[0533] 13-e) The relevant integration efficiency is shown in Table 18. The results show that nox4, nox5, nox8, nox9, nox10, and nox11 can be integrated by themselves with high efficiency, while they cannot integrate with each other.

[0534] Table 18. Integration efficiency of different nox mutations

[0535] Example 14: Simultaneous integration of multiple targets and fragments

[0536] 14-a) Following the construction process of strain BN-1 in 11-a), BN-100 was constructed from strain BN-1, BN-101 was constructed from strain BN-100, and BN-102 was constructed from strain BN-101.

[0537] The difference lies in the use of different primers to replace the original primers at each step, resulting in BN100, BN101, and BN102 having different genotypes. The primers and genotypes used in the construction of different strains are shown in Table 19.

[0538] Table 19. Information on BN100-BN102

[0539] 14-b) Simultaneous integration of three DNA fragments.

[0540] 14-bI) Preparation of Escherichia coli BN-101D electrotransformation competent cells.

[0541] The pW-NC01 plasmid was transferred into the BN-101 strain obtained in Example 14-a) by electroporation and cultured on ampicillin plates at 30°C. Single clones were picked and designated as strain BN-101D. The BN-101D strain was cultured overnight in liquid LB medium, then inoculated into LB medium (containing 50 mg / L ampicillin and a final concentration of 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electroporated competent cells, thus obtaining BN-101D competent cells.

[0542] 14-b-II) Plasmids pKNF-1, pSNF-9, and pCNF-11 were transduced into competent cells BN-101D.

[0543] The plasmids pKNF-1, pSNF-9, and pCNF-11 obtained in Example 12 were electroporated into BN-101D competent cells. After culturing at 30°C for 1 hour, they were spread on solid LB culture dishes containing 50 mg / L streptomycin, 50 mg / L kanamycin, and 17 mg / L chloramphenicol and incubated overnight at 30°C until single colonies emerged from the long plates.

[0544] Identification of strains with integrated three-segment chromosomes (14-b-III)

[0545] Single colonies were picked from the plate and incubated at 30°C for 6 hours in LB medium containing 50 mg / L streptomycin, 50 mg / L kanamycin, and 17 mg / L chloramphenicol. The bacterial culture was then transferred to a PCR reaction system containing three primer pairs: 1pxM-up / P3-R, 1pxB-up / P3-R, and 1ldhA-up / P3-R, for colony PCR amplification. If all bands are approximately 1500 bp in size, integration at all three sites is complete.

[0546] The results showed that when 10 clones were randomly selected, pKNF-1, pSNF-9, and pCNF-11 were all integrated into the BN-101D chromosome, with an integration efficiency of 100%.

[0547] The three exogenous fragments (14-b-IV)EGFP, EYFP, and ECFP are simultaneously integrated into the chromosome.

[0548] The experimental procedure was performed exactly as described in steps 14-bI) to 14-b-III), except that the pKNF-1, pSNF-9, and pCNF-11 plasmids were replaced with pKNF-GFP, pSNF-YFP, and pCNF-CFP, respectively. Specifically, for pKNF-GFP, the following fragment was inserted between the P1 and P2 segments of plasmid pKNF-1: sequences 5'-3' sequentially consisting of SEQ ID NO:143 (Pcpa1 promoter), SEQ ID NO:145 (coding sequence of EGFP), and SEQ ID NO:144 (TrrnB terminator). For pSNF-YFP, the following fragment was inserted between the P1 and P2 segments of plasmid pSNF-9: sequences 5'-3' sequentially consisting of SEQ ID NO:143 (Pcpa1 promoter), SEQ ID NO:146 (coding sequence of EYFP), and SEQ ID NO:144 (TrrnB terminator). pCNF-CFP is formed by inserting the following fragments between the P1 and P2 segments of plasmid pCNF-11: sequences 5'-3' are SEQ ID NO:143 (Pcpa1 promoter), SEQ ID NO:147 (ECFP coding sequence), and SEQ ID NO:144 (TrrnB terminator).

[0549] During the identification process, if the band size is approximately 2500 bp, it indicates successful integration at all three sites simultaneously. Results showed that in a random selection of 10 clones, the three exogenous fragments, EGFP, EYFP, and ECFP, were all correctly integrated into the chromosome, achieving an integration efficiency of 100%.

[0550] 14-c) Simultaneous integration of four DNA fragments.

[0551] 14-cI) to prepare Escherichia coli BN-101D electrotransformation competent cells.

[0552] The pW-NC01 plasmid was transferred into the BN-102 strain obtained in Example 14-a) by electroporation and cultured on ampicillin plates at 30°C. Single clones were picked and designated as strain BN-102D. The BN-102D strain was cultured overnight in liquid LB medium, then inoculated into LB medium (containing 50 mg / L ampicillin and a final concentration of 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electroporated competent cells, thus obtaining BN-102D competent cells.

[0553] 14-c-II) Plasmids pKNF-1, pSNF-9, pCNF-11, and pPNF-4 were transferred into competent cells BN-102D.

[0554] The plasmids pKNF-1, pCNF-9, pSNF-11, and pPNF-4 obtained in Example 12 were electroporated into BN-102D competent cells. After culturing at 30°C for 1 hour, they were spread on solid LB culture dishes containing 50 mg / L streptomycin, 50 mg / L kanamycin, 17 mg / L chloramphenicol, and 50 mg / L ampicillin, and incubated overnight at 30°C until single colonies emerged from the long plates.

[0555] Identification of chromosome integration in strain 14-c-III)

[0556] Single colonies were picked from the plate and incubated at 30°C for 6 hours in LB medium containing 50 mg / L streptomycin, 50 mg / L kanamycin, 17 mg / L chloramphenicol, and 50 mg / L ampicillin. The bacterial culture was then transferred to a PCR reaction system containing four primer pairs: pxM-up / Kan-R, poxB-up / Kan-R, ldhA-up / Kan-R, and pflB-up / Kan-R, for colony PCR amplification. Integration was considered complete when all bands were approximately 1500 bp in size.

[0557] The results showed that when 10 clones were randomly selected, pKNF-1, pSNF-9, pCNF-11, and pPNF-4 were all integrated into chromosome BN-102D, with an integration efficiency of 100%.

[0558] The four exogenous fragments (14-c-IV)EGFP, EYFP, ECFP, and dsRFP) are simultaneously integrated into the chromosome.

[0559] The experimental procedure was performed exactly as described in steps 14-cI) to 14-c-III), except that the pKNF-1, pSNF-9, pCNF-11, and pPNF-4 plasmids were replaced with pKNF-GFP, pSNF-YFP, pCNF-CFP, and pPNF-RFP, respectively. Specifically, pPNF-RFP was created by inserting the following fragment between the P1 and P2 segments of plasmid pPNF-4: sequences 5'-3' sequentially contain SEQ ID NO:143 (Pcpa1 promoter), SEQ ID NO:148 (coding sequence of dsRFP), and SEQ ID NO:144 (TrrnB terminator).

[0560] During the identification process, if all bands are approximately 2500 bp in size, it indicates successful integration at all four sites simultaneously. Results showed that in a random selection of 10 clones, all four exogenous fragments—EGFP, EYFP, ECFP, and dsRFP—were correctly integrated into the chromosome, achieving an integration efficiency of 100%.

[0561] Example 15: CRISPR-mediated multi-site sequential integration

[0562] All the tool plasmids mentioned below were synthesized or spliced ​​from the whole genome. The relevant information is shown in Tables 16 and 17.

[0563] 15-a) Prepare Escherichia coli BW25113 electroporation competent cells, and transfer pW-NC02 plasmid (Table 16) into them by electroporation. Culture them on ampicillin plates at 30°C. Select single clones and designate them as strain BW25113-CW.

[0564] 15-b) CRISPR-mediated sequential integration.

[0565] 15-bI) The BW25113-CW strain was inoculated into LB medium containing 50 mg / L ampicillin and cultured overnight.

[0566] 15-b-II) overnight cultured cells were inoculated into LB medium (containing 50 mg / L ampicillin and 1 μM salicylic acid) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells. The recombinant plasmid pTarget-lpxM-poxB-ldhA (which is obtained by replacing the sequence at positions 227-246 of plasmid pTargetF with the following sequence in sequence: 5'-3' sequentially lpxM sgRNA (SEQ ID NO:128), gRNA backbone (SEQ ID NO:142), P119 promoter (SEQ ID NO:141), poxB sgRNA (SEQ ID NO:130), gRNA backbone (SEQ ID NO:142), P119 promoter (SEQ ID NO:141), ldhA sgRNA (SEQ ID NO:132)) and plasmid pARM-lpxM-poxB-ldhA (5'-3' sequentially p15A (SEQ ID NO:139), ColS (SEQ ID NO:140), Kan (SEQ ID NO:123), L01 fragment (SEQ ID NO:129), P01 fragment (SEQ ID NO:132)) and plasmid pARM-lpxM-poxB-ldhA (5'-3' sequentially p15A (SEQ ID NO:139), ColS (SEQ ID NO:140), Kan (SEQ ID NO:123), L01 fragment (SEQ ID NO:129), P01 fragment (SEQ ID NO:132)) were obtained by replacing the sequence at positions 227-246 of plasmid pTargetF with the following sequence: 5'-3' sequentially lpxM sgRNA (SEQ ID NO:128), gRNA backbone (SEQ ID NO:142), P119 promoter (SEQ ID NO:141), ldhA sgRNA (SEQ ID NO:132)) were obtained. (NO:131) and L02 fragment (SEQ ID NO:133) were electroporated into BW25113-CW competent cells and incubated overnight at 30°C on LB agar plates containing 50 mg / L ampicillin, 50 mg / L streptomycin and 50 mg / L kanamycin.

[0567] 15-b-III) Single colonies were picked from overnight plates and inoculated into LB medium (containing 50 mg / L ampicillin, final concentration 0.5 mM IPTG) and cultured at 30°C for 3 hours. The corresponding liquid culture was then transferred to LB medium (containing 50 mg / L ampicillin, final concentration 0.2% L-arabinose) and cultured at 30°C for 3 hours to prepare electrotransformation competent cells. The plasmids pKNF-1, pSNF-9, and pCNF-11 obtained in Example 12 were electroporated into competent cells. After culturing at 30°C for 2 hours in LB medium containing 50 mg / L streptomycin, 50 mg / L kanamycin, and 17 mg / L chloramphenicol, 100 μL of the culture medium was transferred to 5 mL of LB medium containing 50 mg / L ampicillin and 1 μM tetracycline. The cells were then cultured at 30°C for 2 hours, centrifuged, and spread onto solid LB medium containing 50 mg / L ampicillin. The cells were then incubated overnight at 30°C.

[0568] 15-b-IV) Single colonies were picked and cultured in liquid LB medium containing 50 mg / L ampicillin at 30°C until OD500. 600 =1, transferred to liquid LB medium containing 50 mg / L ampicillin and 50 mg / L kanamycin, and incubated at 30°C for 2 hours. Simultaneously, the liquid culture was amplified by PCR using verification primers lpxM-up / lpxM-down, poxB-up / poxB-down, and ldhA-up / ldhA-down. If the size of all three PCR products was approximately 4000 bp, it was considered a positive clone.

[0569] The results showed that when 10 clones were randomly selected, pKNF-1, pSNF-9, and pCNF-11 were all integrated into the BN-101D chromosome, with an integration efficiency of 100%.

[0570] For strains cultured in 15-b-IV), repeat steps 15-b-II) to 15-b-IV). The difference is that plasmids pSNL-1, pCNL-9, and pKNL-11 are used instead of pKNF-1, pSNF-9, and pCNF-11 for a second round of multi-site integration.

[0571] The results showed that when 10 clones were randomly selected, pSNL-1, pCNL-9, and pKNL-11 were all integrated into the BN-101D chromosome, with an integration efficiency of 100%.

[0572] In Examples 16-28 below, the method of the present invention is described as the "Ein-out method" (Genome Editing through the combination of targeted knock-in and knock-out). Figure 1 is a schematic diagram of the process of integrating large continuous gene fragments in eukaryotic cells using the method of the present invention.

[0573] The experimental procedures in Examples 16-28 below have all been repeatedly verified, and the experimental results are all statistically significant. The specific sequences of each DNA fragment involved in Examples 16-28 below are shown in Table 20, and the sequences of each primer involved in Examples 16-28 below are shown in Table 21.

[0574] Table 20. Sequences of various DNA / RNA fragments

[0575] Table 21, Primer Table

[0576] Example 16, General Experimental Method

[0577] (1) Obtaining DNA fragments

[0578] 16-1-a) PCR amplification method

[0579] Using the synthesized gene as a template, amplification was performed using FastPfu Fly high-fidelity DNA polymerase (TransGen Biotech, catalog number: AP231-21) and specified primers. The PCR products were recovered using the EasyPure DNA Gel Purification Kit (TransGen Biotech, catalog number: EG101-01) to obtain DNA fragments.

[0580] (2) Obtaining plasmids

[0581] 16-2-a) Total Synthesis Method

[0582] Plasmids were obtained using a whole-genome synthesis method.

[0583] 16-2-b) Gibson assembly method

[0584] Using the specified backbone plasmid as a template, amplification was performed using FastPfu Fly high-fidelity DNA polymerase and specified primers. The PCR products were recovered using the EasyPure DNA gel purification kit to obtain plasmid fragments.

[0585] Using the synthesized gene as a template, amplification was performed using FastPfu Fly high-fidelity DNA polymerase and specified primers. The PCR products were recovered using the EasyPure DNA gel purification kit to obtain the inserted DNA fragment.

[0586] The plasmid fragments described above were ligated to the inserted DNA fragment using the Gibson assembly method to obtain the ligation product. The ligation product was electroporated into E. coli DH5α competent cells, clones were selected, and clones capable of amplifying specific fragment sizes were identified using specified primers and sequenced. Plasmids were extracted from correctly identified positive clones to obtain positive plasmids.

[0587] 16-2-c) T4-DNA ligase ligation method

[0588] Using the synthesized gene as a template, amplification was performed using FastPfu Fly high-fidelity DNA polymerase and specified primers. The PCR products were recovered using the EasyPure DNA gel purification kit to obtain the inserted DNA fragment.

[0589] The specified backbone plasmid and insert DNA fragment were treated with a specific restriction endonuclease (NEB, product instructions recommend the digestion conditions), and the treated plasmid fragment and insert DNA fragment were recovered using the EasyPure DNA Gel Purification Kit (TransGen, catalog number: EG101-01).

[0590] The plasmid fragment and the inserted DNA fragment were ligated using T4-DNA ligase (NEB, catalog number: M0202-S) to obtain the ligation product. The ligation product was electrotransformed into E. coli DH5α competent cells, clones were selected, and clones capable of amplifying specific fragment sizes were identified using specified primers and sequenced. Plasmids were extracted from correctly identified positive clones to obtain positive plasmids.

[0591] Information on the DNA fragments involved in this invention is shown in Table 20, and information on the primers used is shown in Table 21.

[0592] Example 17: Transformation and screening of Yersinia lipophila

[0593] (1) All transformations of Yeast lipophila were performed using chemical transformation methods: the transformation was carried out according to the instructions of the Frozen-EZ Yeast Transformation II Kit, catalog number T2001, brand ZYMO RESEARCH.

[0594] (2) Screening of transformants

[0595] 17-2-a) The transformed cells were spread on plates containing the corresponding screening drugs and cultured at 28-30℃ for 2-4 days. The single colonies that grew were the preliminary positive transformants.

[0596] The culture medium formula used is as follows:

[0597] YPD medium: 1L of medium contains 10g yeast extract, 20g peptone, and 20g glucose, pH 6.0. Medium with added selection markers is designated YPD+selection marker.

[0598] Sc medium: 1L of medium contains 6.7g yeast nitrogen basal (YNB), 20g glucose, and 0.69g complete supplement mixture (CSM), pH 6.0. Medium with added selection markers is designated Sc+ selection marker. For auxotrophic selection, the corresponding components are removed, and the medium is designated Sc- selection marker.

[0599] 17-2-b) Commonly Used Screening Markers and Their Working Concentrations

[0600] Hygromycin B resistance marker: Its expression product, hygromycin B phosphotransferase, can induce resistance in cells to hygromycin B. Working concentration: 300-500 μg / mL.

[0601] Noristrine resistance marker: Its expression product, noristrine acetyltransferase, can induce resistance in cells to noristrine. Working concentration: 100-200 μg / mL.

[0602] Uracil auxotroph marker: Orotic nucleoside-5'-phosphate decarboxylase is expressed via the URA3 gene. During positive selection, uracil-deficient strains are reintroduced, allowing transformants to grow on uracil-free media. During negative selection, the added 5-fluoroorotic acid (5-FOA) is converted to 5-fluorouridine monophosphate (5-FUMP), preventing transformants containing this gene from growing. Working concentration of 5-FOA: 0.8-1 mg / mL.

[0603] Leucine auxotroph marker: Leucine-deficient strains were replenished by using the LEU gene-encoded α-isopropylmalate isomerase, enabling transformants to grow on leucine-free basic media.

[0604] Some examples of tags include:

[0605] YPD+hyg medium: YPD medium containing hygromycin; Sc-ura medium: Complete medium without ura; Sc+FOA medium: Synthetic complete medium containing 5-FOA; Sc-leu medium: Synthetic complete medium without leu; YPD+ntc medium: YPD medium containing nouricin.

[0606] Example 18: Transformation and Screening of Mammalian Cells

[0607] (1) Cell preparation

[0608] 18-1-a) Preparation of CHO cells:

[0609] One day before transformation, 293T cells or CHO cells in the logarithmic growth phase were digested with trypsin, resuspended and counted, and then seeded at an appropriate density in culture plates or dishes and cultured in a 37°C, 5% CO2 incubator to achieve 70%-90% confluence at the time of transformation.

[0610] 18-1-b) Preparation of primary cells such as T cells:

[0611] See subsequent examples for details, depending on the source of the cells.

[0612] (2) Conversion Method

[0613] Any one of these can be selected in subsequent embodiments.

[0614] 18-2-a) Liposome conversion (using Lipofectamine 3000 as an example):

[0615] It is suitable for 293T cells, CHO cells, and some easily transfected primary cells.

[0616] Step 1: Complex preparation

[0617] In serum-free media such as Opti-MEM, plasmid DNA was mixed with P3000 TM Enhancer mixture (Solution A). In another tube, mix Lipofectamine 3000 transfection reagent with Opti-MEM (Solution B). After incubating at room temperature for 5 minutes, mix Solution A with Solution B and let stand at room temperature for 15-20 minutes to form a DNA-liposome complex.

[0618] Step 2: Conversion

[0619] Add the above complex dropwise to the cells that have been replaced with fresh culture medium (which may contain serum), and gently shake the culture plate to mix it.

[0620] Step 3: Cultivation

[0621] Incubate the cells at 37°C in a 5% CO2 incubator. After 4-6 hours, replace the medium with complete medium and continue culturing for another 24-72 hours before screening.

[0622] 18-2-b) Electroconversion method:

[0623] Suitable for CHO cells, difficult-to-transfect primary cells (such as T cells and neuronal stem cells), and suspension culture of 293T cells.

[0624] Step 1: Preparation of cell suspension

[0625] Digest adherent cells with trypsin or resuspend the cells in buffer, then collect by centrifugation. Wash the cells thoroughly 1-2 times with pre-chilled electroporation buffer (such as PBS or a dedicated cell electroporation solution), and finally resuspend them in an appropriate amount of electroporation buffer, adding plasmid DNA or CRISPR ribonucleoprotein complex.

[0626] Step 2: Electric shock

[0627] Transfer the cell-DNA mixture to a pre-chilled electroporation cuvette. Using an electroporator, set optimized parameters according to cell type (e.g., for CHO cells: voltage 250-350V, capacitance 950μF; for primary T cells: voltage 1300-1500V, pulse width 10-30ms). Perform electroporation.

[0628] Step 3: Recovery

[0629] Immediately after electroporation, add preheated complete culture medium to the electroporation cuvette and gently mix by pipetting. Transfer the cell suspension to a culture plate and incubate at 37°C in a 5% CO2 incubator. Replace with fresh culture medium after 24 hours, continue culturing, and then perform screening.

[0630] (3) Screening of transformants

[0631] 18-3-a) After transformation, cells begin to show significant cell death after culturing in drug-containing medium for approximately 24-48 hours. Continue culturing for 5-14 days, changing the drug-containing selection medium every 2-3 days, until resistant clones are formed. For limiting dilution clones, cells can be diluted to a low seed density to facilitate single-clone selection.

[0632] 18-3-b) Commonly Used Screening Markers and Their Working Concentrations

[0633] Puromycin resistance marker: Its expression product, puromycin-N-acetyltransferase, can induce cell resistance to puromycin.

[0634] Working concentration: 1-10 μg / mL (the specific concentration needs to be determined based on the cell line and the killing curve. For example, 1-2 μg / mL is commonly used for 293T cells, 2-5 μg / mL is commonly used for CHO cells, and the concentration may be lower for primary cells).

[0635] G418 (Genzyme) resistance marker: Its expression product, neomycin phosphotransferase, can induce cell resistance to G418.

[0636] Working concentration: 200-1000 μg / mL (Vast differences between different cell lines, for example, 400-800 μg / mL is commonly used for CHO cells, and 300-500 μg / mL is commonly used for 293T cells, and the killing concentration needs to be determined in advance).

[0637] Hygromycin B resistance marker: Its expression product, hygromycin B phosphotransferase, can induce cell resistance to hygromycin B.

[0638] Working concentration: 50-400 μg / mL (e.g., 100-200 μg / mL is commonly used for mammalian cells).

[0639] Example 19: Transformation and Screening of Plant Cells

[0640] (1) Preparation of the receptor system

[0641] 19-1-a) Induction and culture of callus tissue:

[0642] Immature embryos, leaves, or stem segments of plants (such as rice, Arabidopsis thaliana, and tobacco) were used as explants and inoculated onto a solid induction medium containing 2,4-dichlorophenoxyacetic acid. The explants were cultured at 25±2℃ in the dark or with low light for 2-4 weeks to induce the formation of loose, pale yellow embryogenic callus.

[0643] 19-1-b) Establishment of suspension cell lines:

[0644] The embryogenic callus tissue was transferred to liquid culture medium and cultured on a shaker at 25±2℃ and 100-130 rpm. Subculture was performed every 5-7 days, and cell lines with rapid growth and uniform particle size were selected for transformation.

[0645] 19-1-c) Preparation of the bladed disc:

[0646] Take healthy, tender leaves from sterile tissue culture seedlings and cut them into leaf discs with a diameter of about 0.5 cm using a sterile punch or scalpel, which are then used for transformation.

[0647] (2) Conversion Method

[0648] 19-2-a) Agrobacterium-mediated transformation:

[0649] It is suitable for most dicotyledonous and monocotyledonous plants (such as rice, tobacco, and Arabidopsis thaliana).

[0650] Step 1: Agrobacterium culture

[0651] Agrobacterium strains containing recombinant binary vectors were inoculated into YEP liquid medium containing the corresponding antibiotics and cultured at 28°C and 200 rpm until mid-log growth.

[0652] Step 2: Co-culture

[0653] Collect the bacterial cells and resuspend them in an equal volume of liquid co-culture medium to adjust the bacterial concentration. Immerse the prepared plant recipient material in the bacterial solution for several minutes, remove it, blot off excess bacterial solution, transfer it to a co-culture medium lined with filter paper, and co-culture at 25°C in the dark for 2-3 days.

[0654] Step 3: Sterilization and Screening

[0655] The co-cultured recipient material was transferred to an antibiotic-containing sterile medium to inhibit Agrobacterium overgrowth. Subsequently, the material was transferred to a differentiation medium containing screening drugs for screening of resistant callus.

[0656] 19-2-b) Gene gun transformation:

[0657] It is suitable for plants that are not easily infected by Agrobacterium, especially important cereal crops (such as corn and wheat).

[0658] Step 1: Micro-elastic wrapping

[0659] Plasmid DNA is encapsulated on the surface of a microelastic carrier such as gold powder or tungsten powder.

[0660] Step 2: Bombardment

[0661] The prepared receptor material is placed in the sample chamber of the gene gun, and under certain helium pressure, vacuum degree and bombardment distance, the micro-projectile is driven to be injected into the receptor cell at high speed.

[0662] Step 3: Recovery and Filtering

[0663] After being bombarded, the recipient material was cultured on a medium without screening pressure for 1-2 days before being transferred to a medium containing screening drugs for screening of resistant callus.

[0664] (3) Screening and regeneration of transformants

[0665] 19-3-a) Screening for resistant callus:

[0666] The transformed recipient material was cultured on a medium containing the corresponding screening drug at 25±2℃ under light conditions. Subcultures were performed every 2 weeks, and screening was continued for 4-8 weeks until vigorous resistant callus tissue was formed.

[0667] 19-3-b) Plant regeneration:

[0668] The resistant callus tissue was transferred to a differentiation medium containing cytokinin to induce shoot differentiation. When the shoots grew to 2-3 cm in height, they were cut off and transferred to a rooting medium containing auxin to induce rooting, forming complete regenerated plants.

[0669] 19-3-c) Commonly used screening markers and their working concentrations

[0670] Hygromycin B resistance marker: Its expression product, hygromycin B phosphotransferase, can induce plant cells to develop resistance to hygromycin B.

[0671] Working concentration: 10-50 mg / L (for use in culture media).

[0672] Kanamycin / Genomycin resistance markers: their expression product, neomycin phosphotransferase, can induce resistance in plant cells to kanamycin or G418.

[0673] Working concentration: 50-100 mg / L (for use in culture media).

[0674] Glufosinate resistance marker: Its expression product, phosphinic acid acetyltransferase, can induce plant cells to develop resistance to glufosinate.

[0675] Working concentration: 2-10 mg / L (for use in culture media).

[0676] Example 20: Chromosome editing of Yersinia lipophila strain using the Ein-out method

[0677] The plasmids and DNA fragments were prepared in accordance with Example 1.

[0678] The transformation and screening of Yeast lipophila were carried out according to Example 2.

[0679] (1) Using Yersinia lipolyticis Po1f as the starting strain, an LN fragment containing the lox66-nox sequence was integrated into the POX5 position to obtain strain P01. The specific operation is as follows:

[0680] 20-1-a) Targeting a specific coding region of the POX5 gene, the following sgRNA target sequence was designed: 5'-GCTGGGTCACGGATCTAACG-3' (SEQ ID NO: 314). The whole-gene plasmid pCRISPR-Sg1 was synthesized, with the following structural composition: 5'-P tRNA-gly -sgRNAPX5-P TEF1 -yCas9-T cyc1 -URA3-YlARS-ColE-Amp-3' (The complete sequence of the plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0681] 20-1-b) The following DNA fragment P1LN was artificially synthesized: 5'-POX5up-lox66-nox-POX5down-3' (the sequence of the DNA fragment P1LN is obtained by sequentially linking the above elements from the 5' end to the 3' end; the sequences of each element are shown in Table 20). Using this fragment as a template and P-1 / P-2 as primers (sequences are shown in Table 21), homologous recombination fragments were prepared by PCR amplification.

[0682] 20-1-c) The pCRISPR-Sg1 plasmid and homologous recombination fragment obtained above were transformed into the target strain using chemical transformation. Transformants were picked and cultured overnight at 30°C in Scura liquid medium. The genome of the transformants was extracted and verified by PCR using primers N-1 / N-2 (sequences shown in Table 21). A positive result was indicated by the amplification of a target band of approximately 2000 bp. The verified strain was streaked onto Sc+FOA plates and cultured overnight at 30°C. Single colonies were transferred to YPD liquid medium and cultured overnight at 30°C, which yielded the strain that lost the pCRISPR-Sg1 plasmid. Thus, strain P01, which integrates the lox66-nox sequence at the POX5 site on the genome, was obtained.

[0683] (2) Integrating the MVA1 fragment into Yersinia lipophila using the “Ein-out” method

[0684] 20-2-a) pNigri-Y1 plasmid transformation. The pNigri-Y1 plasmid was synthesized entirely artificially, and its structural composition is as follows: 5'-P TEF1 -Nigri-T cyc1-URA3-YlARS-ColE-Amp-3' (The complete sequence of the plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; see Table 20 for the sequence of each element). The pNigri-Y1 plasmid was transformed into the above-mentioned strain P01 by chemical transformation. Transformants were picked and inoculated into Sc-ura liquid medium and cultured overnight at 30°C to obtain strain P01-Nigri.

[0685] Transformation with the 20-2-b)pNL-MVA1 plasmid. The plasmid contains the MVA1 fragment (P...). TEF1 -ERG10-T cyc1 -P TEF1 -ERG13-T cyc1 -P TEF1 -HMG2-T cyc1 -P TEF1 -ERG12-T cyc1 The pNL-MVA1 plasmid was synthesized entirely artificially, and its structural composition is as follows: 5'-nox-HygR1-ColE-Amp-lox71-P TEF1 -ERG10-T cyc1 -P TEF1 -ERG13-T cyc1 -P TEF1 -HMG2-T cyc1 -P TEF1 -ERG12-T cyc1 -loxF1-3' (The complete sequence of the plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; the sequences of each element are shown in Table 20). The pNL-MVA1 plasmid was transformed into the above-mentioned strain P01-Nigri using chemical transformation. The plasmid was then plated on Sc+hyg+FOA plates.

[0686] 20-2-c) Transformants were picked and cultured in YPD+hyg liquid medium. The genome was extracted and verified by PCR using primer N-1 / MVA-1 (sequence shown in Table 21). The 5800bp target band was amplified as an integration positive clone, and sequencing was used to verify that the knock-in fragment sequence was correct.

[0687] When evaluating integration efficiency, the positive integration rate is defined as the ratio of the number of clones identified as having positive integration to the number of randomly selected clones. The positive colony number (PN / μg DNA) is the number of positive colonies (PN) obtained by converting a unit mass of circular DNA molecules added.

[0688] 20-2-d) Deletion of HygR1 selection marker: The pCre-Y1 plasmid was introduced via chemical transformation. The pCre-Y1 plasmid was entirely synthetically produced, and its structural composition is as follows: 5'-PTEF1 -Cre-T cyc1 -URA3-YlARS-ColE-Amp-3' (The complete sequence of this plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; the sequences of each element are shown in Table 20). Transformants were picked and inoculated into Sc-ura liquid medium and cultured overnight at 30°C. They were then streaked onto Sc+FOA plates. Single colonies were cultured in YPD liquid medium, and the genome was extracted. The deletion of the HygR1 selection marker was verified by PCR using primers N-1 / MVA-1 (sequences shown in Table 21). A 1500bp target band indicating the removal of the selection marker was amplified, and sequencing confirmed the correctness of the knock-in fragment. This constitutes the clone with the HygR1 selection marker removed.

[0689] 20-2-e) To verify the efficiency of Nigri-mediated integration, the following control was set up in processes 20-2-a) to 20-2-c): pCre-Y1 plasmid was used instead of pNigri-Y1 plasmid; pCre-Y1 plasmid was introduced by chemical transformation to test whether the circular DNA molecule could recombine with the lox66 site on the genome under the action of Cre enzyme.

[0690] Results analysis (Table 22):

[0691] In experiments 20-2-b) and 20-2-c), a large number of transformant clones grew from the pNL-MVA1 plasmid after transformation. Twenty clones were randomly selected for identification, and all were positive, resulting in a 100% integration rate. The number of positive clones was (1.23 ± 0.10) × 10⁻⁶. 4 PN / μg DNA. In 20-2-e), pNigri-Y1 was replaced with pCre-Y1, and no transformants grew on the plate. Sequencing of positive clones from 20-2-c) and 20-2-d) showed that no mutations occurred in the knock-in sequence of all positive clones, and only one nox site remained on the genome after the selection marker was deleted, allowing for further precise integration. These results indicate that the method provided by this invention can achieve high-efficiency editing in Yersinia lipophila.

[0692] Table 22. Efficiency of MVA1 fragment integration at the POX5 position in the Yersinia lipolyticis genome

[0693] Example 21: Sequential chromosome editing of *Yarrowia lipophila* strain using the Ein-out method

[0694] The plasmids and DNA fragments were prepared in accordance with Example 16.

[0695] The transformation and screening of Yeast lipophila were carried out according to Example 17.

[0696] (1) The first round of editing was completed in accordance with the process in Example 20. The strain was identified by the process described in Example 20. The positive clone with the HygR1 resistance gene eliminated was named P02.

[0697] (2) The second round of editing was completed exactly as described in Example 20, except that strain P02 was used instead of P01 to transform the pNigri-Y1 plasmid to obtain strain P02-Nigri; and pNF-MVA2 plasmid was used instead of pNL-MVA1. The pNF-MVA2 plasmid was entirely artificially synthesized, and its structural composition is as follows: 5'-nox-HygR1-ColE-Amp-loxF2-P TEF1 -ERG8-T cyc1 -P TEF1 -ERG19-T cyc1 -P TEF1 -IDI-T cyc1 -P TEF1 -ERG20-T cyc1 -lox66-3' (The complete sequence of the plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; the sequences of each element are shown in Table 20). The pNF-MVA2 plasmid was transformed into the above-mentioned strain P02-Nigri using chemical transformation. The plasmid was plated on YPD+hyg plates. The strain obtained by transforming P02 was identified as described in Example 5, except that primer N-7 / MVA-2 (sequence shown in Table 21) was used instead of primer N-1 / MVA-1. After correct integration, the PCR amplification target band of the positive clone was 5100 bp, and after eliminating the resistance gene, the size was 500 bp. The positive clone with the resistance gene eliminated was named P03.

[0698] (3) The third round of editing was completed exactly as described in Example 20, except that: strain P03 was used instead of P01 to transform the pNigri-Y1 plasmid to obtain strain P03-Nigri; and pNL-MVA3 plasmid was used instead of pNL-MVA1. The pNL-MVA3 plasmid was entirely artificially synthesized, and its structural composition is as follows: 5'-nox-HygR1-ColE-Amp-lox71-P TEF1 -CRTE-T cyc1 -P TEF1 -CRTB-T cyc1 -P TEF1 -CRTI-T cyc1-loxF1-3' (The complete sequence of the plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; the sequences of each element are shown in Table 20). The pNF-MVA3 plasmid was transformed into the above-mentioned strain P11-Nigri using chemical transformation. The plasmid was plated on YPD+hyg plates. The strain obtained by transforming P03 was identified as described in Example 20, except that primer N-8 / MVA-3 (sequence shown in Table 21) was used instead of primer N-1 / MVA-1. After correct integration, the PCR amplification target band of the positive clone was 5100 bp, and after eliminating the resistance gene, the size was 500 bp. The positive clone with the resistance gene eliminated was named P04.

[0699] Results analysis:

[0700] PCR identification confirmed that chromosome integration in *Yarrowia lipolyticis* using the Ein-out method of this invention achieved 100% integration efficiency in the first, second, and third rounds. The number of positive clones in the first, second, and third rounds were (1.23 ± 0.10) × 10⁻⁶, respectively. 4 PN / μg DNA, (5.78±0.32)×10 3 PN / μg DNA, (1.01±0.09)×10 3 PN / g DNA (Figure 2). These results demonstrate that the method provided by this invention can achieve highly efficient continuous editing in Yersinia lipophila.

[0701] Example 22: Large-segment chromosome editing of *Yarrowia lipophila* strain using the Ein-out method.

[0702] The plasmids and DNA fragments were prepared in accordance with Example 16.

[0703] The transformation and screening of Yeast lipophila were carried out according to Example 17.

[0704] The editing was performed exactly as described in Example 20, except that pNL-AV plasmid was used instead of pNL-MVA1. The pNL-AV plasmid was entirely artificially synthesized, and its structural composition is as follows: 5'-nox-HygR1-ColE-Amp-lox71-P TEF1 -AV1-T cyc1-loxF1-3' (The complete sequence of the plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; the sequences of each element are shown in Table 20). The insert fragment is approximately 60 kb in size. The pNL-AV plasmid was transformed into the above-mentioned strain P01-Nigri using chemical transformation. The plasmid was plated on YPD-hyg plates. Clones on the plates were identified as described in Example 5, except that primer N-1 / AV-1 (sequence shown in Table 21) was used instead of primer N-1 / MVA-1. After correct integration, the PCR amplification target band of the positive clone was 6800 bp. After eliminating the resistance gene, the size was 2200 bp. The positive strain was named P05. The number of positive clones was determined.

[0705] Results analysis:

[0706] PCR identification confirmed that chromosome integration in *Yarrowia lipolyticis* using the EIN-OUT method of this invention achieved a 100% integration efficiency. The number of positive clones was (8.72±1.01)×10⁻⁶. 2 PN / μg DNA. The above results demonstrate that the method provided by this invention can achieve highly efficient integration of large fragments in *Yarrowia lipophila*.

[0707] Example 23: Multi-site editing of chromosomes in *Yarrowia lipophila* strains using the Ein-out method.

[0708] The plasmids and DNA fragments were prepared in accordance with Example 16.

[0709] The transformation and screening of Yeast lipophila were carried out according to Example 17.

[0710] The editing was completed exactly as described in Example 20, except that:

[0711] The P11 strain was used to transform the pNigri-Y1 plasmid instead of the P01 strain to obtain the P11-Nigri strain. The POX5, POX1, and POX3 gene loci of the P11 strain were inserted into lox66-nox, lox66-nox4, and lox66-nox5 loci, respectively. The construction steps of the P11 strain were performed according to Example 20. The insertion of lox66-nox into the POX5 locus was exactly the same as in Example 20. The difference in the insertion of lox66-nox4 into the POX1 locus was: the pCRISPR-Sg1 plasmid was replaced with the pCRISPR-Sg2 plasmid; the DNA fragment P2LN was used instead of the DNA fragment P1LN when preparing the homologous recombination fragment; primers P-3 / P-4 (sequences shown in Table 21) were used instead of P-1 / P-2; and primers N-3 / N-4 (sequences shown in Table 21) were used instead of N-1 / N-2 for PCR verification, resulting in a positive result with an amplified target band of approximately 2000 bp. The pCRISPR-Sg2 plasmid has a 5'-P structure. tRNA-gly -sgRNAPX1-P TEF1 -yCas9-T cyc1 -URA3-YlARS-ColE-Amp-3' (The complete sequence of the plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element). The structure of the DNA fragment P2LN is 5'-POX1up-lox66-nox4-POX1down-3' (The sequence of the DNA fragment P2LN is obtained by sequentially linking the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element). The operational differences in inserting the POX3 site into lox66-nox5 are as follows: The pCRISPR-Sg1 plasmid is replaced with the pCRISPR-Sg3 plasmid; when preparing the homologous recombination fragment, the DNA fragment P3LN is replaced with the DNA fragment P1LN, and primers P-5 / P-6 (sequences shown in Table 21) are used instead of P-1 / P-2; during PCR verification, primers N-5 / N-6 (sequences shown in Table 21) are used instead of N-1 / N-2, and a positive result is achieved when approximately 2000 bp of the target band is amplified. The pCRISPR-Sg3 plasmid has a 5'-P... tRNA-gly -sgRNAPX3-P TEF1 -yCas9-T cyc1 -URA3-YlARS-ColE-Amp-3' (The complete sequence of the plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element). The structure of the DNA fragment P3LN is 5'-POX3up-lox66-nox5-POX3down-3' (The sequence of the DNA fragment P3LN is obtained by sequentially linking the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0712] The transformation process involved the simultaneous transformation of pNL-MVA1 plasmid (see Example 20), pNLG-MVA2 plasmid, and pNLL-MVA3 plasmid. pNLG-MVA2 and pNLL-MVA3 plasmids were entirely synthetically produced. The structural composition of pNLG-MVA2 plasmid is 5'-nox4-LEU-ColE-Amp-lox71-P. TEF1 -ERG10-T cyc1 -P TEF1 -ERG13-T cyc1 -P TEF1 -HMG2-T cyc1 -P TEF1 -ERG12-T cyc1 -loxF1-3'. The structure of the pNLL-MVA3 plasmid is 5'-nox5-NTC-ColE-Amp-lox71-P TEF1 -CRTE-T cyc1 -P TEF1 -CRTB-T cyc1 -P TEF1 -CRTI-T cyc1 -loxF1-3'. The plasmids pNL-MVA1, pNLG-MVA2, and pNLL-MVA3 were transformed into the above-mentioned strain P11-Nigri using chemical transformation. The plasmids were plated on Sc-leu+hyg+ntc plates. The obtained strain was identified using primers N-1 / MVA-1, N-3 / MVA-2, and N-5 / MVA-3. Clones amplified by primers N-1 / MVA-1, N-3 / MVA-2, and N-5 / MVA-3 with target fragments of 5800bp, 5900bp, and 5000bp respectively were considered positive clones. The number of positive clones was determined. The positive clone with all three resistance marker genes eliminated was named P12.

[0713] Results analysis:

[0714] PCR identification confirmed that the Ein-out method of this invention achieved 100% integration efficiency into multiple chromosomal sites in *Yarrowia lipolyticis*. The number of positive clones was (2.55±0.31)×10⁻⁶. 2 PN / μg DNA. The above results demonstrate that the method provided by this invention can efficiently perform simultaneous chromosome editing at at least three sites in *Yarrowia lipolyticis*.

[0715] Example 24: Constructing a protein expression library using CHO cells

[0716] The plasmids and DNA fragments were prepared in accordance with Example 16.

[0717] The transformation and screening of CHO cells were carried out according to Example 17.

[0718] (1) Construction of CHO-H11-LN cell line

[0719] In the chromosomal sequence of CHO cells, the H11 site can efficiently and stably express exogenous proteins. Therefore, a sequence containing the lox66-nox exogenous gene fragment was inserted into the H11 site of CHO cells to serve as the chassis cell for constructing a stable exogenous protein expression library. The specific operation is as follows:

[0720] 24-1-a) Target design and sgRNA expression plasmid construction

[0721] Designed sgRNA target sequence H11-sgRNA1: 5'-TATACACTTGAGCCAGTAGTGGG-3' (SEQ ID NO:315).

[0722] The commercially available CRISPR / Cas9 vector pSpCas9(BB)-2A-GFP(PX458)(Addgene plasmid#48138) was used as the backbone plasmid, hereinafter referred to as PX458.

[0723] Primers designed: H11-sgRNA1-F / H11-sgRNA1-R (sequences are shown in Table 21).

[0724] After mixing the H11-sgRNA1-F / R primers, the mixture was annealed at 95°C for 5 min to obtain the H11-sgRNA fragment. The vector pX458 was digested with BbsI enzyme and then ligated to the H11-sgRNA fragment according to the procedure in Example 16 to construct a plasmid named pX458-H11-sgRNA. The structural description of pX458-H11-sgRNA is as follows: a recombinant plasmid obtained by inserting the H11-sgRNA1 target sequence into the BbsI site of pX458.

[0725] 24-1-b) Preparation of NL fragment targeting plasmid pH11-NL

[0726] The pH11-NL plasmid was synthesized entirely artificially. Its structural composition is 5'-ColE-Amp-H11up-lox66-G418-nox-H11down-3' (the complete sequence of the plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. The sequence of each element is shown in Table 20).

[0727] 24-1-c) Cell transfection and selection

[0728] Using the CHO-K1 cell line, pX458-H11-sgRNA and pH11-NL plasmids were transfected via electroporation. Forty-eight hours after transfection, the medium was replaced with complete medium containing G418 for selection, continuing for 48-72 hours to kill untransfected cells. Surviving cells were digested, counted, and diluted to a very low density, then seeded into 96-well plates, ensuring approximately 0.5 cells per well. Cells were cultured for approximately 10-14 days, with regular observation and labeling of clonal colonies formed from single cells. Genomic DNA was extracted from well-grown single colonies. Successful integration of the NL fragment was detected using identification primers N-9 / N-10 (sequences shown in Table 21). A band of approximately 2300 bp was observed upon successful NL integration. The positive cell line was named CHO-H11-LN.

[0729] (2) Constructing a CHO cell library containing an EF1α promoter library using EIN-OUT

[0730] The 24-2-a)pNL1-PEF-GFP plasmid was synthesized entirely artificially, and its structural composition is 5'-nox-P CMV -NLS1-Cre-IRES-EBFP2-PolyA_SG-ColE-Amp-lox71-P EF1α -loxF1-EGFP-IRES-HygR2-PolyA_SG-3' (The complete sequence of this plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0731] 24-2-b) The mutant library fragment was amplified by PCR using pNL1-PEF-GFP plasmid as template and primers P-7 / P-8 (sequences are shown in Table 21) with error-prone PCR kit (Shanghai Zeye Company, catalog number ZY6351PD).

[0732] The vector fragment was amplified by PCR using pNL1-PEF-GFP plasmid as template and primers V-1 / V-2 (sequences are shown in Table 21).

[0733] Following the Gibson method in Example 16, the vector fragment and the mutant library fragment were ligated to obtain the plasmid library pLib-EF.

[0734] The 24-2-c)pNigri-C1 plasmid was synthesized entirely artificially, and its structural composition is 5'-ColE-Amp-P tet-on -NLS1-Nigri-IRES-mCherry-PolyA_SG-3' (The complete sequence of the plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0735] 24-2-d) Seed CHO-H11-LN cells into suitable multi-well plates or culture flasks, and transfect when the density reaches 70-80%. The pNigri-C1 plasmid and pLib-EF plasmid library are co-introduced into the cells using electroporation. 48 hours after transfection, the medium is replaced with selection medium containing hygromycin (300 μg / mL) and doxycycline (1 μg / mL). After 2-3 days of selection, the medium is replaced with selection medium containing hygromycin (300 μg / mL) and cultured for 3-4 days to kill untransfected cells that have integrated the fragment. The surviving cell pool is the cell population with the EF1α promoter library integrated into the chromosome. Cells exhibiting only green fluorescence but no red or blue fluorescence are sorted using flow cytometry. The selected cells should be those whose fragment has been integrated into the chromosome, and whose redundant sequences (including one nox site after recombination) have been eliminated.

[0736] 24-4-c) Identification and screening of EF1α promoter libraries

[0737] CHO cell lines containing the EF1α promoter library were cultured in 10cm dishes. A portion of the cells were harvested and screened using flow cytometry for cells exhibiting different green fluorescence signals. These cells were then sorted into 96-well plates, one cell per well. The 96-well plates were incubated at 37°C until 80% confluence. The cells were then digested and divided into three groups: 1. Transferred to 24-well plates for further culture and preservation; 2. Green fluorescence intensity was verified using flow cytometry; 3. Transferred to a new 96-well plate (PCR plate) for genomic DNA extraction. Genomic DNA was extracted from 30-50 cell lines with the required green fluorescence intensity (appropriate GFP expression level), and PCR was used to assess the efficiency of Ein-out specific integration. During the identification process, we used multiple primer pairs for PCR amplification to achieve accurate typing, as shown in Figure 3(A): (1) Primers N-11 and N-18, located upstream and downstream of the integration site, were paired with primers N-14 and N-16 inside the integrated DNA fragment for amplification to confirm integration-positive cells; (2) Primer pairs N-11 and N-18 were used for amplification to distinguish between integration-negative cells and heterozygous cells; (3) Primers N-11 and N-12 were used for amplification to identify cells with incomplete integration (although integration has occurred, the upstream nox sequence has not been eliminated); (4) Primer pairs N-13 / N-14, N-15 / N-17, and N-12 / N-16 were used for amplification to determine whether there were randomly inserted cells. The PCR identification results of cells with different genotypes are shown in Table 23. The mutated EF1α promoter sequence was obtained by PCR amplification using primers N-9 / N-10 and then sequencing. The sequences of the primers mentioned above are shown in Table 21.

[0738] Table 23. PCR identification results of cells with different genotypes Note: The genotype determination rules are as follows: 1. Determination of integration positive, integration negative, heterozygous, and incomplete integration: The amplification results of the 7 pairs of primers in the corresponding column must be completely matched (" / " indicates a non-critical indicator and is not included in the determination); if the results do not match, it is classified as "unidentified". 2. Determination of random integration: Two conditions must be met simultaneously: (1) The amplification results of primers N-11 / N-14 and N-16 / N-18 are both "unable to amplify"; (2) At least one of the following amplification results must be met: primers N-11 / N-18 amplify to obtain a product of 1800bp; primers N-13 / N-14 amplify to obtain a product of 2900bp; primers N-15 / N-17 amplify to obtain a product of 1900bp; primers N-12 / N-16 amplify to obtain a product of 3300bp. If not met, it is classified as "unidentified".

[0739] As shown in Figure 4, the percentage of cells with positive integration in the CHO expression library (the percentage of cells with positive integration identified by PCR) was above 73.45 ± 4.45%. The method provided in this invention for constructing expression libraries (EF1α promoter libraries) in CHO cells has significant advantages: (1) it minimizes the probability of non-specific integration such as random integration, greatly improving library quality; (2) after library construction, flow cytometry can be used for rapid quality control and screening, shortening the work cycle; (3) the integration site retains a single nox site, allowing for rapid re-editing of the next round of genes without additional genome modification.

[0740] Example 25: Integration and screening of continuous chromosome libraries in CHO cells using the Ein-out method. The preparation of the plasmids and DNA fragments was carried out in accordance with Example 16.

[0741] The transformation and screening of CHO cells were carried out according to Example 17.

[0742] (1) First round of library integration and screening based on EF1α promoter library

[0743] The method is the same as the process in Example 24. In this process, different cells sorted by flow cytometry were analyzed for the integration of P fragments. EF1α The sequences contain different mutation sites (EF1α promoter library). Based on requirements, stable expression cells with weak green fluorescence are screened by flow cytometry to obtain suitable EF1α promoter mutants, which will facilitate subsequent affinity screening. The optimal cell line selected is named CHO-EF1-WO3, where the integrated fragment contains P... EF1α The sequence is P EF1α-M3. The CHO-EF1-WO3 cell line was used as the cell line for the next round of chromosome integration. In this cell line, using the method described in Example 24, the following sequence was integrated into the H11 position of the CHO-K1 cell line chromosome: 5'-lox72-P EF1α -M3-loxF1-EGFP-IRES-HygR2-PolyA_SG-nox-3' (a sequence formed by sequentially linking the elements; see Table 20 for the sequence of each element).

[0744] (2) Second round of library integration and screening based on CD19 mutant libraries

[0745] The 25-2-a)pNL2-CD19 plasmid was synthesized entirely artificially, and its structural composition is 5'-nox-P CMV -NLS1-Cre-PolyA_SG-ColE-Amp-loxF2-CD19-PolyA_SG-lox66-PuroR(reverse)-3' (The complete sequence of the plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0746] 25-2-b) The mutant library fragment was amplified by PCR using pNL2-CD19 plasmid as template and primers P-9 / P-10 (sequences are shown in Table 21) with error-prone PCR kit (Shanghai Zeye Company, catalog number ZY6351PD).

[0747] The vector fragment was amplified by PCR using pNL2-CD19 plasmid as template and primers V-1 / V-3 (sequences are shown in Table 21).

[0748] Following the Gibson method in Example 16, the vector fragment and the mutant library fragment were ligated to obtain the plasmid library pLib-CD19.

[0749] 25-2-c) Seed CHO-EF1-WO3 cells into suitable multi-well plates or culture flasks, and transfect when the density reaches 70-80%. Co-introduce the pNigri-C1 plasmid and pLib-CD19 plasmid library into the cells using electroporation. 48 hours after transfection, replace the medium with selection medium containing puromycin (2 μg / mL). Continue selection for 7-10 days to kill untransfected cells that have integrated the fragment. The surviving cell pool is the cell population containing the CD19 mutant library integrated into the chromosome. Use flow cytometry to sort cells that do not show red or green fluorescent signals. The selected cells should be those where the CD19 mutant fragment has been integrated into the chromosome, and where redundant sequences (including one of the nox sites after recombination) have been eliminated.

[0750] 25-2-d) Identification and screening of CD19 mutant libraries

[0751] Cell banks containing the CD19 mutant library were cultured in 10cm dishes. A portion of the cells were incubated with anti-CD19 antibody and labeled secondary antibody. Cells showing significantly increased affinity for the anti-CD19 antibody were screened using flow cytometry and sorted into 96-well plates, one cell per well. The 96-well plates were incubated at 37°C until 80% confluence. The cells were then digested and divided into three groups: 1. Transferred to 24-well plates for further culture and preservation; 2. Flow cytometry was used to verify affinity for the anti-CD19 antibody; 3. Transferred to a new 96-well plate (PCR plate) for genomic DNA extraction. Genomic DNA was extracted from 20-40 cell lines, and PCR was used to determine the efficiency of Ein-out specific integration. Multiple primer pairs were used for PCR amplification during the identification process to achieve accurate typing, as shown in Figure 3(B): (1) Primers N-11 and N-18, located upstream and downstream of the integration site, were paired with primers N-19 and N-21 inside the integrated DNA fragment for amplification to confirm integration-positive cells; (2) Primer pairs N-11 and N-18 were used for amplification to distinguish between integration-negative cells and heterozygous cells; (3) Primers N-11 and N-12 were used for amplification to identify cells with incomplete integration (although integration has occurred, the upstream nox sequence has not been eliminated); (4) Amplification was performed using primer pairs N-16 / N-19, N-20 / N-22, and N-12 / N-21 to determine whether there were randomly inserted cells. The PCR identification results for cells with different genotypes are shown in Table 24. The sequences of the primers mentioned above are shown in Table 21.

[0752] Table 24. PCR identification results of cells with different genotypes Note: The genotype determination rules are as follows: 1. Determination of integration positive, integration negative, heterozygous, and incomplete integration: The amplification results of the 7 pairs of primers in the corresponding column must be completely matched (" / " indicates a non-critical indicator and is not included in the determination); if the results do not match, it is classified as "unidentified". 2. Determination of random integration: Two conditions must be met simultaneously: (1) The amplification results of primers N-11 / N-19 and N-18 / N-21 are both "unable to amplify"; (2) At least one of the following amplification results must be met: primers N-11 / N-18 amplify a 4000bp product; primers N-20 / N-22 amplify a 1800bp product; primers N-12 / N-21 amplify a 2000bp product. If not met, it is classified as "unidentified".

[0753] The results showed that 98% of the sorted cells could amplify the specific integration target bands (positive target bands amplified at N-11 / N-19 and N-18 / N-21), and there were no integration-negative cells or incompletely integrated cells. The percentage of integration-positive cells (the percentage of integration-positive cells identified by PCR out of the total cells) reached over 79.44 ± 8.22%, as shown in Figure 4. These results indicate that the method provided by this invention can achieve rapid, continuous, and highly efficient library integration on the genome.

[0754] Example 26: Construction of a stable CAR-T expression cell line

[0755] The plasmids and DNA fragments were prepared in accordance with Example 16.

[0756] The transformation and screening of T cells were carried out in accordance with Example 17.

[0757] (1) Isolation and activation of peripheral blood T cells from patients

[0758] 26-1-a) Peripheral blood single cell isolation: Peripheral blood was collected from cancer patients in heparinized tubes. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation. Cells were resuspended in PBS and counted.

[0759] 26-1-b) T Cell Sorting and Enrichment: Uncultured, high-purity T cells were isolated from PBMCs using a commercially available T cell negative selection magnetic bead kit. The purity (CD3+) of the sorted cells was assessed by flow cytometry. + The cell percentage should be >95%. Resuspend the cells in T cell culture medium containing 10% human AB serum (i.e., serum derived from human AB blood type antibodies, without anti-A or anti-B blood type antibodies, for lymphocyte culture) and 300 IU / mL IL-2.

[0760] 26-1-c) T cell activation: T cells were activated at a rate of 1×10⁻⁶. 6 T cells were seeded at a density of 1 cell / mL in culture plates pre-coated with anti-human CD3 and anti-human CD28 antibodies. 100 IU / mL of recombinant human IL-2 was added. The cells were incubated at 37°C, 5% CO2 for approximately 48 hours to activate T cells.

[0761] (2) Establish CAR stable expression cell lines using the “Ein-out” method

[0762] 26-2-a) Preparation of pegRNA

[0763] For the CCR5 gene locus, pegRNAs based on twinPE-guided editing were designed, and 2'-O-methyl modified pegRNAs, CNpegRNA1 and CNpegRNA2 (sequences are shown in Table 20), were synthesized by Shanghai Sangon Biotech.

[0764] 26-2-b) Preparation of Cas9 nickase-reverse transcriptase fusion protein (PE2) mRNA

[0765] The commercial plasmid pCMV-PE2 (addgene, #132775) was linearized using PmeI restriction endonuclease (NEB, catalog number R0560L), and then the mRNA was synthesized using the NEB HiScribe T7 ARCA kit. After the reaction, the mRNA was treated with DNase I (NEB, M0303L) and then purified using a Monarch RNA purification column (NEB T2040L).

[0766] 26-2-c) Preparation of Nigri mRNA

[0767] The pCMV-Nigri plasmid was synthesized entirely artificially, specifically by replacing the Cas9 reading frame in the pCMV-PE2 plasmid with the Nigri gene (sequence shown in Table 20). The preparation method of Nigri mRNA was completely consistent with the method in 26-2-b), except that the pCMV-PE2 plasmid was replaced with the pCMV-Nigri plasmid.

[0768] 26-2-d) The target plasmid pNox-CAR plasmid was synthesized entirely artificially, and its structural composition is 5'-nox-P CMV -NLS1-Cre-IRES-EGFP-PolyA_SG-ColE-Amp-lox71-CAR(reverse)-loxF1-P EF1α (Reverse)-3' (The complete sequence of the plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; see Table 20 for the sequence of each element). CAR is the gene encoding the chimeric antibody used in CAR-T cell therapy for multiple myeloma.

[0769] Integration of 26-2-e)pNox-CAR: Take 5×10 5Cells activated in step (1) were resuspended in B1mix electroporation buffer, and 2 μg of mRNA prepared in step 26-2-b), 2 μg of mRNA prepared in step 26-2-c), 100 pmol CNpegRNA1 / 2, and 5 μg of plasmid pNox-CAR were added and mixed thoroughly. Electroporation was performed using a Lonza 4D Nucleofector system. Immediately after electroporation, 80 μL of preheated cell culture medium was added to the electroporation cuvette, and the cuvette was placed in an incubator and incubated for 10 min. The liquid was then transferred to a 96-well plate for further culture. 24 hours after transfection, the cells were centrifuged to change the medium. 72 hours after transfection, the cells were collected for genomic DNA extraction and subsequent analysis.

[0770] Screening and gene integration analysis of 26-2-f) positive cell lines

[0771] Integration efficiency was assessed using high-throughput sequencing on the Illumina sequencing platform (Novaseq 6000). DNA extracted from T cells using a genomic DNA extraction kit (QIAGEN#69504) was quantified via Nanodrop or Qubit. The target genomic region was first amplified using forward and reverse primer sequences (see Table 21). Then, Illumina sequencing adapters and barcodes were introduced for a second round of amplification, followed by library construction and sequencing. Editing efficiency was analyzed using the specialized tool CRISPResso2. Integration positive efficiency (%) = (total number of edited reads / total number of valid aligned reads) × 100%. T cells were sorted by flow cytometry. Cells were treated with recombinant CD319 protein and mouse anti-human CD319 flow cytometry antibody (BD Biosciences, catalog number 750832). Cells showing positive antibody signals but negative green fluorescence signals were screened, and single viable cells were directly aliquoted into 96-well plates for culture. Subsequent CAR-T function validation was then performed.

[0772] Results analysis:

[0773] In the 26-2-e) process, the integration positivity rate based on Illumina sequencing reached 2.8 ± 0.54%. The method provided by this invention leverages the advantages of small enzyme molecular weight and high catalytic efficiency, enabling genome integration in primary human cells using a relatively simple DNA delivery method. The integrated cells were sorted by flow cytometry and subsequently validated, successfully obtaining a series of stably expressing cell lines; sequencing confirmed that the CAR gene cluster could accurately integrate into the target chromosomal site.

[0774] Example 27: Gene insertion into rice protoplasts using the Ein-out method

[0775] The plasmids and DNA fragments were prepared in accordance with Example 16.

[0776] The transformation and screening of T cells were carried out in accordance with Example 17.

[0777] (1) Preparation of rice protoplasts

[0778] Healthy 3-4 leaf stage rice seedlings of Zhonghua 1 were selected and enzymatically hydrolyzed at 28°C with shaking for 4-5 hours in the dark using a hydrolysate containing 1.5% cellulase R-10, 0.75% cleavage enzyme R-10, and 0.5% pectinase Y-23 (formulation: 0.6M mannitol, 10mM MES pH 5.7, 10mM CaCl2). The reaction product was filtered through a 200-mesh nylon screen, and protoplasts were collected by differential centrifugation at 100×g for 5 minutes. The protoplasts were then washed sequentially with pre-cooled W5 solution (formulation: 154mM NaCl, 125mM CaCl2) in an ice bath and resuspended in MMg solution (formulation: 0.6M mannitol, 15mM MgCl2) for purification.

[0779] (2) Utilizing Ein-out to integrate target genes

[0780] 27-2-a) Preparation of pCP-RNA-Nigri plasmid

[0781] The pCP-RNA-Nigri plasmid was synthesized entirely artificially, and its structural composition is 5'-ColE-Amp-P. Os_ubi -NLS2-SpCas9(H840A)-Linker1-MLVRT-NLS2-T NOS -P Os_ubi -NLS2-Nigri-T NOS -P Os_U3 -PpegRNA1-P Os_U3 -PpegRNA2-3' (The complete sequence of this plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; the sequences of each element are shown in Table 20). Among them, PpegRNA1 contains lox66, and PpegRNA2 contains nox.

[0782] 27-2-b) Preparation of the targeting vector pNox-GFP plasmid

[0783] The pNox-GFP plasmid was synthesized entirely artificially, and its structural composition is 5'-nox-P Os_ubi -NLS2-Cre-T NOS -P Os_ubi -mCherry-T NOS -ColE-Amp-lox71-P Os_ubi -EGFP-T NOS -POs_ubi -Zm25860-T NOS -loxF1-3' (The complete sequence of the plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0784] 27-2-c) Transfection and screening of integrative plasmids

[0785] High-quality plasmids were prepared using the Wizard Plus Midipreps DNA purification system. 5 μg of each plasmid prepared in 27-2-a) and 27-2-b) were mixed and used to transform the protoplasts prepared in Example 27(1) using a polyethylene glycol-mediated transformation method. After 24 hours of transformation, red and green fluorescence were detected by flow cytometry to preliminarily assess the transformation status; cells exhibiting both red and green fluorescence were considered successfully transformed and integrated with the plasmid. The transformed protoplasts were cultured at 26°C for another 72 hours, and the cells were collected by centrifugation. A portion of these cells were analyzed by flow cytometry; cells exhibiting only green fluorescence and no red fluorescence were considered positive cells that had successfully integrated and eliminated one of the nox sites. The remaining cells were used for subsequent genomic DNA extraction. DNA extraction and positivity rate assessment followed the method in 26-2-f).

[0786] In process 27-2-c), the integration positivity rate based on Illumina sequencing reached 82.8 ± 10.2%. The method provided by this invention avoids the drawbacks of other integrases, such as low integration direction efficiency, large molecular weight, and lack of continuous editing capability at the integration site. It also offers advantages such as flexible editing design. It shows promising application prospects in plant genome integration.

[0787] Example 28: Integrating drought-resistant genes into maize plant chromosomes using the "Ein-out" method.

[0788] The plasmids and DNA fragments were prepared in accordance with Example 16.

[0789] The conversion and screening of corn were carried out according to Example 17.

[0790] (1) Plasmid preparation

[0791] The pCP2-RNA plasmid was synthesized entirely artificially, and its structural composition is 5'-ColE-Amp-P. Zm_ubi -NLS2-SpCas9(H840A)-Linker1-MLVRT-NLS2-T NOS -P Zm_U3 -PpegRNA3-P Zm_U3-PpegRNA4-3' (The complete sequence of this plasmid is obtained by sequentially linking the above elements from the 5' end to the 3' end; the sequences of each element are shown in Table 20). Among them, PpegRNA3 contains lox66, and PpegRNA4 contains nox.

[0792] The pZm-HPT plasmid was synthesized entirely artificially, and its structural composition is 5'-P Zm_ubi -HPT-T NOS -ColE-Amp-3' (The complete sequence of the plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0793] The pM-Nigri plasmid was synthesized entirely artificially, and its structural composition is 5'-ColE-Amp-P. Zm_ubi -NLS2-Nigri-T NOS -3' (The complete sequence of the plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0794] The pNox-Zm plasmid was synthesized entirely artificially, and its structural composition is 5'-nox-P. Zm_ubi -NLS2-Cre-T NOS -P Zm_ubi -mCherry-T NOS -ColE-Amp-lox71-BAR-P Zm_ubi -Zm25860-T NOS -loxF1-3' (The complete sequence of the plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. See Table 20 for the sequence of each element).

[0795] (2) Construction of the Z01 strain

[0796] Immature embryos of the maize inbred line B104 were used as recipient material. Agrobacterium-mediated transformation was used to introduce pCP2-RNA plasmid and pZm-HPT plasmid into embryogenic callus. Multiple rounds of selection were performed on hygromycin-containing selection medium to obtain resistant callus. Genomic DNA was extracted from the resistant callus. Genome sequencing confirmed the successful and precise introduction of the nox site at the target site, without any other additional indel mutations. The successfully validated line was named Z01.

[0797] (3) Integration of large-segment stress-resistance genes

[0798] Z01 callus tissue was spread on a hypertonic medium and pre-dried. The pM-Nigri and pNox-Zm plasmids prepared in Example 28(1) were transfected into the callus tissue using a gene gun transfection method. The callus tissue was transferred to recovery medium and incubated in the dark at 26°C for 16-24 hours. Subsequently, the tissue was transferred to a subculture medium containing glufosinate for screening and proliferation of resistant callus tissue. The genome sequence of the integrated plant was identified using genome sequencing.

[0799] The results showed that in T1 plants, a large fragment of the stress resistance gene was precisely integrated as a single copy at the pre-defined safe harbor site, without any exogenous selection markers or recombinase genes. This indicates that the method of the present invention can be effectively applied to genome integration in plants such as maize.

[0800] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Industrial applicability

[0801] This invention enables rapid, continuous, multi-site integration of the host cell genome through simple molecular biology operations, while also efficiently completing large-fragment integration operations. Furthermore, this invention can leverage CRISPR technology to mediate the aforementioned genome integration process, significantly improving the accuracy and efficiency of integration.

Claims

1. A kit characterised in that: The complete product is either a complete product 1 for integrating the genome of a host cell or a complete product 2 for continuously integrating the genome of a host cell. The complete set of products 1 includes (A1), (A3) and (A4) as follows; or, the complete set of products 1 includes (A2), (A3) and (A4) as follows; The complete set of products 2 includes the following (A1), (A2), (A3) and (A4); (A1) Circular vector 1; the circular vector 1 is composed of the following elements in the order from 5' to 3': loxF1, nox, replication origin site, resistance gene, lox71, P3, P1, P2; (A2) Circular vector 2; the circular vector 2 is composed of the following elements in the order from 5' to 3': lox66, nox, replication origin site, resistance gene, loxF2, P3, P1, P2; (A3) Circular vector 3; the circular vector 3 is used to express Nigri recombinase and Cre recombinase; (A4) Site-specific knock-in tool; the site-specific knock-in tool is capable of knocking in a specific DNA fragment at the integration site of the host cell's genome; the specific DNA fragment is composed of lox66 and nox or loxF1 and nox; P1 and P2 are adapter sequences, and the position between P1 and P2 is used to insert the DNA fragment to be integrated. P3 is a universal primer sequence.

2. The kit of claim 1, wherein: In both the annular carrier 1 and the annular carrier 2, the replication initiation site is oriR6k.

3. A kit according to claim 1 or 2, characterised in that: The nucleotide sequence of loxF1 is SEQ ID NO:1; and / or The nucleotide sequence of the NOX is SEQ ID NO:2; and / or The nucleotide sequence of oriR6k is SEQ ID NO:3; and / or The nucleotide sequence of lox71 is SEQ ID NO:5; and / or The nucleotide sequence of P3 is SEQ ID NO:6; and / or The nucleotide sequence of P1 is SEQ ID NO:7; and / or The nucleotide sequence of P2 is SEQ ID NO:8; and / or The nucleotide sequence of lox66 is SEQ ID NO:9; and / or The nucleotide sequence of loxF2 is SEQ ID NO:

10.

4. The kit of claim 1, wherein: The circular vector 3 contains an expression cassette 1 for expressing the Cre recombinase, an expression cassette 2 for expressing the Nigri recombinase, and a replication origin site.

5. The kit of claim 4, wherein: In expression cassette 1, the promoter for initiating transcription of the gene encoding the Cre recombinase is ParaBAD, and the termination sequence is TrrnB; and / or, in expression cassette 2, the promoter for initiating transcription of the gene encoding the Nigri recombinase is Ptet, and the termination sequence is TrrnB; and / or, the replication initiation site is RK2s, wherein RK2s is a temperature-sensitive RK2 replication initiation site.

6. The kit of claim 5, wherein: The circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, the resistance gene, and RK2s.

7. A kit according to claim 5 or 6, characterised in that: The nucleotide sequence of ParaBAD is SEQ ID NO:16; and / or The nucleotide sequence of the gene encoding the Cre recombinase is SEQ ID NO:13; and / or The nucleotide sequence of the TrrnB is SEQ ID NO:20; and / or The nucleotide sequence of the Ptet is SEQ ID NO:19; and / or The nucleotide sequence of the gene encoding the Nigri recombinase is SEQ ID NO:15; and / or The nucleotide sequence of the RK2s is SEQ ID NO:

21.

8. The kit of claim 1, wherein: The targeted knock-in tool consists of the following components 1)-3): 1) a targeting vector containing a coding sequence of sgRNA targeting the integration site in the host cell genome; 2) a homologous recombination fragment containing an upstream homologous arm, the specific DNA fragment, and a downstream homologous arm from the 5' to 3' ends; the upstream homologous arm is a fragment located upstream of the integration site in the host cell genome; the downstream homologous arm is a fragment located downstream of the integration site in the host cell genome; and 3) an expression vector for expressing Cas protein and λ-red element.

9. The kit of claim 8, wherein: The targeted knock-in tool is composed of 1) and 2); and the ring carrier 3 also contains an expression cassette 3 for expressing the Cas protein and an expression cassette 4 for expressing the λ-red element.

10. The kit of claim 9, wherein: In expression cassette 3, the promoter for initiating transcription of the gene encoding the Cas protein is Psal, and the termination sequence is TrrnB; and / or, in expression cassette 4, the promoter for initiating transcription of the coding sequence of the λ-red element is Psal.

11. The kit of claim 10, wherein: The circular vector 3 is composed of the following elements in the order from 5' to 3': Psal, the gene encoding the Cas protein, TrrnB, Psal, exo-bet-gam gene, TλtL3 coding sequence, ColRNA coding sequence, ParaBAD, the gene encoding the Cre recombinase, TrrnB, Ptet, the gene encoding the Nigri recombinase, TrrnB, resistance gene, and RK2s.

12. A kit according to any one of claims 1 to 11, characterised in that: The host cell can be a bacterium, fungus, animal cell, or plant cell.

13. The kit of claim 12, wherein: The host cell is Escherichia coli.

14. The kit of claim 13, wherein: The integration site is located at the lpxM gene position in E. coli, and the integration is the replacement of the lpxM gene; the coding sequence of the sgRNA is SEQ ID NO:25, and the sequence of the homologous recombination fragment is SEQ ID NO:

26.

15. A method for integrating the genome of a host cell, implemented using the kit product 1 described in any one of claims 1-14; the method comprising the following steps: (a1) Using the aforementioned knock-in tool, the specific DNA fragment is knocked into the integration site in the genome of the host cell; (a2) Connect the adapter sequences to both ends of the DNA fragment to be integrated, i.e., connect one end to P1 and the other end to P2; use homologous recombination to clone the DNA fragment to be integrated into the circular vector 1 or the circular vector 2 between P1 and P2 to obtain a recombinant vector; (a3) The circular carrier 3 is introduced into the host cells treated with (a1); (a4) The recombinant vector obtained in (a2) is introduced into the host cell treated in (a3), and after culturing and screening, recombinant cells with one copy of the DNA fragment to be integrated knocked into the integration site are obtained.

16. A method for sequential integration of the genome of a host cell, implemented using the kit product 2 described in any one of claims 1-14; the method comprising the following steps: (b1) Using the aforementioned knock-in tool, the specific DNA fragment is knocked into the integration site in the genome of the host cell; (b2) Connect the adapter sequence to both ends of all the DNA fragments to be integrated, i.e., connect one end to P1 and the other end to P2; after numbering the DNA fragments to be integrated, clone the DNA fragments to be integrated with odd numbers into the circular vector 1 between P1 and P2 using homologous recombination to obtain several recombinant vectors 1, each carrying one DNA fragment to be integrated; clone the DNA fragments to be integrated with even numbers into the circular vector 2 between P1 and P2 using homologous recombination to obtain several recombinant vectors 2, each carrying one DNA fragment to be integrated; (b3) The circular carrier 3 is introduced into the host cells treated with (b1); (b4) Introduce either the recombinant vector 1 or the recombinant vector 2 obtained in (b2) into the host cell treated in (b3), and after culturing and screening, obtain a recombinant cell with one copy of the first DNA fragment to be integrated knocked into the integration site, denoted as recombinant cell 1; then introduce the other recombinant vector 2 or the recombinant vector 1 obtained in (b2) into the recombinant cell 1, and after culturing and screening, obtain a recombinant cell with one copy of the first DNA fragment to be integrated and one copy of the second DNA fragment to be integrated knocked into the integration site, denoted as recombinant cell 2; in this step, if the first introduction is the recombinant vector 1, then the second introduction is the recombinant vector 2; if the first introduction is the recombinant vector 2, then the second introduction is the recombinant vector 1. (b5) Referring to (b4), in the alternating order of introducing the recombinant vector 1 and the recombinant vector 2, all the remaining recombinant vector 1 and all the remaining recombinant vector 2 are sequentially introduced into the recombinant cells obtained in the previous step. The final recombinant cells are the recombinant cells in which one copy of all the DNA fragments to be integrated is knocked into the integration site at the same time.

17. A reagent kit, characterized in that: The kit is kit 1 for constructing a target plasmid for large-fragment integration of the host cell genome or kit 2 for constructing a target plasmid for continuous large-fragment integration of the host cell genome. The kit 1 comprises (B1) and (B3) as follows; or, the kit 1 comprises (B2) and (B3) as follows; The kit 2 includes the following (B1), (B2) and (B3); (B1) Circular vector 1; the circular vector B1 is composed of the following elements in the order from 5' to 3': loxF1, replication origin site ColE, resistance gene 1, loxF, nox, replication origin site oriSC101, resistance gene 2, lox71, P3, P1, P2. (B2) Circular vector 2; the circular vector B2 is composed of the following elements in the order from 5' to 3': lox66, replication origin site ColE, resistance gene 1, loxP, nox, replication origin site oriSC101, resistance gene 2, loxF2, P3, P1, P2; (B3) Site knock-in tool 1; the site knock-in tool 1 is capable of knocking in a specific DNA fragment 1 at a certain site in the genome of the host cell; the specific DNA fragment 1 contains an expression cassette 1 for expressing Cre recombinase and an expression cassette 2 for expressing repA1 protein; P1 and P2 are adapter sequences, and the position between P1 and P2 is used to insert the DNA fragment to be integrated. P3 is a universal primer sequence.

18. The kit according to claim 17, characterized in that: The complete product also contains the host cell.

19. The kit according to claim 17, characterized in that: The nucleotide sequence of loxF1 is SEQ ID NO:1; and / or The nucleotide sequence of the replication origin site ColE is SEQ ID NO:31; and / or The nucleotide sequence of loxF is SEQ ID NO:34; and / or The nucleotide sequence of the NOX is SEQ ID NO:2; and / or The nucleotide sequence of the replication origin site oriSC101 is SEQ ID NO:33; and / or The nucleotide sequence of lox71 is SEQ ID NO:5; and / or The nucleotide sequence of P3 is SEQ ID NO:6; and / or The nucleotide sequence of P1 is SEQ ID NO:7; and / or The nucleotide sequence of P2 is SEQ ID NO:8; and / or The nucleotide sequence of lox66 is SEQ ID NO:9; and / or The nucleotide sequence of the loxP is SEQ ID NO:35; and / or The nucleotide sequence of loxF2 is SEQ ID NO:

10.

20. The kit according to claim 17, characterized in that: The fixed-point tapping tool 1 consists of the following 1)-3) composition: 1) A targeting vector containing a coding sequence for an sgRNA targeting a specific site in the genome of the host cell; 2) A homologous recombination fragment containing an upstream homologous arm, the specific DNA fragment 1, and a downstream homologous arm from the 5' to the 3' end; the upstream homologous arm is a fragment located upstream of the specific site in the genome of the host cell; the downstream homologous arm is a fragment located downstream of the specific site in the genome of the host cell; 3) An expression vector for expressing Cas protein and λ-red element.

21. The kit according to claim 17, characterized in that: In expression cassette 1, the promoter for initiating transcription of the gene encoding the Cre recombinase is ParaBAD, and the termination sequence is TrrnB; and / or, in expression cassette 2, the promoter for initiating transcription of the gene encoding the repA1 protein is Ptrc, and the termination sequence is TrrnB.

22. The kit according to claim 21, characterized in that: The specific DNA fragment 1 is composed of the following elements in 5' to 3' order: ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptrc, the gene encoding repA1 protein, and TrrnB.

23. The kit according to claim 21 or 22, characterized in that: The nucleotide sequence of ParaBAD is SEQ ID NO:16; and / or The nucleotide sequence of the gene encoding the Cre recombinase is SEQ ID NO:13; and / or The nucleotide sequence of the TrrnB is SEQ ID NO:20; and / or The nucleotide sequence of the Ptrc is SEQ ID NO:29; and / or The nucleotide sequence of the gene encoding the repA1 protein is SEQ ID NO:

30.

24. The kit according to any one of claims 17-23, characterized in that: The host cell can be a bacterium, fungus, animal cell, or plant cell.

25. The kit according to claim 24, characterized in that: The host cell is Escherichia coli.

26. The reagent kit according to claim 25, characterized in that: The specified site is located at the lpxM gene position in E. coli, and the knock-in is the replacement of the lpxM gene; the coding sequence of the sgRNA is SEQ ID NO:25; the nucleotide sequence of the upstream homologous arm is SEQ ID NO:27; and / or, the nucleotide sequence of the downstream homologous arm is SEQ ID NO:

28.

27. A method for constructing a targeting plasmid for large-fragment or sequential integration of the genome of a host cell, implemented using the kit described in any one of claims 17-26; the method comprising the following steps: (c1) Using the site-specific knock-in tool 1, the specific DNA fragment 1 is knocked into a specific site in the genome of the host cell; (c2) Connect the adapter sequences to both ends of the DNA fragment to be integrated, i.e., connect one end to P1 and the other end to P2; clone the DNA fragment to be integrated into the circular vector 1 between P1 and P2 using homologous recombination to obtain recombinant vector 1; and / or clone the DNA fragment to be integrated into the circular vector 2 between P1 and P2 using homologous recombination to obtain recombinant vector 2; (c3) The recombinant vector 1 and / or the recombinant vector 2 obtained in (c2) are introduced into or separately introduced into the host cells treated in (c1). After screening and induction of protein expression, the Cre recombinase mediates the recombination of loxF1 and loxF on the recombinant vector 1 to eliminate the replication origin site ColE on the recombinant vector 1, and the resulting plasmid is the target plasmid 1; and / or, the Cre recombinase mediates the recombination of lox66 and loxP on the recombinant vector 2 to eliminate the replication origin site ColE on the recombinant vector 2, and the resulting plasmid is the target plasmid 2.

28. A complete set of products, characterized in that: The complete set of products is either a complete set of products 3 for integrating large fragments of the host cell genome or a complete set of products 4 for continuously integrating large fragments of the host cell genome. The complete set of products 3 includes the following (C1), (C3) and (C4); The complete set of products 4 includes the following (C2), (C3) and (C4); (C1) The kit 1 as described in any one of claims 17-26, or the target plasmid 1 or the target plasmid 2 constructed using the method described in claim 27; (C2) The kit 2 as described in any one of claims 17-26, or the target plasmid 1 and the target plasmid 2 constructed using the method described in claim 27; (C3) Circular vector 3; the circular vector B3 is used to express Nigri recombinase and Cre recombinase; (C4) Knock-in tool 2; the knock-in tool 2 is capable of knocking in a specific DNA fragment 2 at the integration site of the host cell's genome; the specific DNA fragment 2 is composed of lox66 and nox or loxF1 and nox.

29. The complete set of products according to claim 28, characterized in that: The circular vector 3 contains an expression cassette 3 for expressing the Cre recombinase, an expression cassette 4 for expressing the Nigri recombinase, and a replication initiation site.

30. The complete set of products according to claim 29, characterized in that: In expression cassette 3, the promoter for initiating transcription of the gene encoding the Cre recombinase is ParaBAD, and the termination sequence is TrrnB; and / or, in expression cassette 4, the promoter for initiating transcription of the gene encoding the Nigri recombinase is Ptet, and the termination sequence is TrrnB; and / or, the replication initiation site is RK2s, wherein RK2s is a temperature-sensitive RK2 replication initiation site.

31. The complete set of products according to claim 30, characterized in that: The circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, the resistance gene, and RK2s.

32. The complete set of products according to claim 30 or 31, characterized in that: The nucleotide sequence of ParaBAD is SEQ ID NO:16; and / or The nucleotide sequence of the gene encoding the Cre recombinase is SEQ ID NO:13; and / or The nucleotide sequence of the TrrnB is SEQ ID NO:20; and / or The nucleotide sequence of the Ptet is SEQ ID NO:19; and / or The nucleotide sequence of the gene encoding the Nigri recombinase is SEQ ID NO:15; and / or The nucleotide sequence of the RK2s is SEQ ID NO:

21.

33. The complete set of products according to claim 28, characterized in that: The fixed-point tapping tool 2 consists of the following 1)-3): 1) A targeting vector containing a coding sequence of sgRNA targeting the integration site in the genome of the host cell; 2) A homologous recombination fragment containing an upstream homologous arm, the specific DNA fragment 2, and a downstream homologous arm from the 5' to 3' ends; the upstream homologous arm is a fragment located upstream of the integration site in the genome of the host cell; the downstream homologous arm is a fragment located downstream of the integration site in the genome of the host cell; 3) An expression vector for expressing Cas protein and λ-red element.

34. The complete set of products according to any one of claims 28-33, characterized in that: The host cell can be a bacterium, fungus, animal cell, or plant cell.

35. The complete set of products according to claim 34, characterized in that: The host cell is Escherichia coli.

36. The complete set of products according to claim 35, characterized in that: The integration site is located at the lpxM gene position in E. coli, and the integration is the replacement of the lpxM gene; the coding sequence of the sgRNA is SEQ ID NO:25, and the sequence of the homologous recombination fragment is SEQ ID NO:

26.

37. A method for integrating large fragments of the host cell genome, implemented using the kit product 3 described in any one of claims 28-36; the method comprising the following steps: (d1) Using the site-specific knock-in tool 2, the specific DNA fragment 2 is knocked into the integration site in the genome of the host cell; (d2) The circular carrier 3 is introduced into the host cells treated with (d1); (d3) The target targeting plasmid 1 or the target targeting plasmid 2 of claim 27 is introduced into the host cell after treatment (d2), and recombinant cells with one copy of the DNA fragment to be integrated knocked into the integration site are obtained after culture and screening.

38. A method for the continuous integration of large fragments of a host cell's genome, implemented using the kit product 4 described in any one of claims 28-36; the method comprising the following steps: (e1) The specific DNA fragment 1 is knocked into a specific site in the genome of the host cell using the site-specific knock-in tool 1; (e2) Connect the adapter sequence to both ends of all the DNA fragments to be integrated, i.e., connect one end to P1 and the other end to P2; after numbering the DNA fragments to be integrated, clone the DNA fragments to be integrated with odd numbers into the circular vector 1 between P1 and P2 using homologous recombination to obtain several recombinant vectors 1, each carrying one DNA fragment to be integrated; clone the DNA fragments to be integrated with even numbers into the circular vector 2 between P1 and P2 using homologous recombination to obtain several recombinant vectors 2, each carrying one DNA fragment to be integrated; (e3) Several recombinant vectors 1 and several recombinant vectors 2 obtained in (e2) are respectively introduced into the host cells treated in (e1). After screening and induction of protein expression, the Cre recombinase mediates the recombination of loxF1 and loxF on the recombinant vector 1 to eliminate the replication origin site ColE on the recombinant vector 1, thereby obtaining several target plasmids 1; the Cre recombinase mediates the recombination of lox66 and loxP on the recombinant vector 2 to eliminate the replication origin site ColE on the recombinant vector 2, thereby obtaining several target plasmids 2. (e4) Using the site-specific knock-in tool 2, the specific DNA fragment 2 is knocked into the integration site in the genome of the host cell; (e5) The circular carrier 3 is introduced into the host cells after treatment (e4); (e6) Introduce either the target plasmid 1 or the target plasmid 2 obtained in (e3) into the host cell treated in (e5), and after culturing and screening, obtain a recombinant cell with one copy of the first DNA fragment to be integrated knocked into the integration site, denoted as recombinant cell 1; Introduce the other target plasmid 2 or the target plasmid 1 obtained in (e3) into the recombinant cell 1, and after culturing and screening, obtain a recombinant cell with one copy of the first DNA fragment to be integrated and one copy of the second DNA fragment to be integrated knocked into the integration site, denoted as recombinant cell 2; In this step, if the first introduction is the target plasmid 1, then the second introduction is the target plasmid 2; if the first introduction is the target plasmid 2, then the second introduction is the target plasmid 1. (e7) Referring to (e6), in the alternating order of introducing the target plasmid 1 and the target plasmid 2, all the remaining target plasmid 1 and all the remaining target plasmid 2 are sequentially introduced into the recombinant cells obtained in the previous step. The final recombinant cells are the recombinant cells in which one copy of all the DNA fragments to be integrated is knocked into the integration site at the same time.

39. Any of the following applications: (D1) The use of the complete product according to any one of claims 1-14 in the integration or continuous integration of the genome of a host cell; (D2) The use of the kit according to any one of claims 17-26 in constructing a target plasmid required for large-fragment or sequential integration of the genome of a host cell; (D3) The use of the complete product according to any one of claims 28-36 in the large-fragment integration or continuous integration of the genome of a host cell.

40. A complete set of DNA molecules consists of all or some of the following: (E1) A DNA molecule with the nucleotide sequence SEQ ID NO:5 is denoted as nox4; (E2) A DNA molecule with the nucleotide sequence SEQ ID NO:6 is denoted as nox5; (E3) A DNA molecule with the nucleotide sequence SEQ ID NO:9 is denoted as nox8; (E4) A DNA molecule with the nucleotide sequence SEQ ID NO:10 is denoted as nox9; (E5) A DNA molecule with the nucleotide sequence SEQ ID NO:11 is denoted as nox10; (E6) A DNA molecule with the nucleotide sequence SEQ ID NO:12 is denoted as nox11; (E7) A DNA molecule with the nucleotide sequence SEQ ID NO:1 is denoted as nox.

41. A DNA fragment or plasmid containing the complete set of DNA molecules as described in claim 40.

42. A complete set of products, characterized in that: The complete product set is either a complete product set 5 for multi-target and multi-fragment integration of the host cell genome or a complete product set 6 for continuous multi-target and multi-fragment integration of the host cell genome. The complete set of products 5 includes (F1), (F3) and (F4) as follows; or, the complete set of products 5 includes (F2), (F3) and (F4) as follows; The complete set of products 6 includes the following (F1), (F2), (F3) and (F4); (F1) Several circular vectors 1; the circular vectors 1 are composed of the following elements in the order from 5' to 3': loxF1, noxM, replication origin site, resistance gene, lox71, P3, P1, P2; (F2) Several circular vectors 2; the circular vectors 2 are composed of the following elements in the order from 5' to 3': lox66, noxM, replication origin site, resistance gene, loxF2, P3, P1, P2; (F3) Circular vector 3; the circular vector 3 is used to express Nigri recombinase and Cre recombinase; (F4) Several site-specific knock-in tools; the site-specific knock-in tools are capable of knocking in a specific DNA fragment into the genome of the host cell at the integration site; the specific DNA fragment is composed of lox66 and noxM or loxF1 and noxM; In (F1), (F2) and (F4), noxM is nox, nox4, nox5, nox8, nox9, nox10 or nox11 as described in claim 40; The noxM differs in different annular carriers 1; The noxM differs in different annular carriers 2; The noxM varies depending on the specific pinpoint input tool used. Furthermore, for the same target point, the noxM in the annular carrier 1 and / or the annular carrier 2 is consistent with the noxM in the pinpoint knock-in tool; P1 and P2 are adapter sequences, the position between P1 and P2 is used to insert the DNA fragment to be integrated, and P3 is a universal primer sequence; The number of items is 2-7.

43. The complete set of products according to claim 42, characterized in that: In both the annular carrier 1 and the annular carrier 2, the replication initiation site is R6k.

44. The complete set of products according to claim 42 or 43, characterized in that: The nucleotide sequence of loxF1 is SEQ ID NO:13; and / or The nucleotide sequence of R6k is SEQ ID NO:14; and / or The nucleotide sequence of lox71 is SEQ ID NO:15; and / or The nucleotide sequence of P3 is SEQ ID NO:16; and / or The nucleotide sequence of P1 is SEQ ID NO:17; and / or The nucleotide sequence of P2 is SEQ ID NO:18; and / or The nucleotide sequence of lox66 is SEQ ID NO:19; and / or The nucleotide sequence of loxF2 is SEQ ID NO:

20.

45. The complete set of products according to claim 42, characterized in that: The circular vector 3 contains an expression cassette 1 for expressing the Cre recombinase, an expression cassette 2 for expressing the Nigri recombinase, and a replication origin site.

46. ​​The complete set of products according to claim 45, characterized in that: In expression cassette 1, the promoter for initiating transcription of the gene encoding the Cre recombinase is ParaBAD, and the termination sequence is TrrnB; and / or, in expression cassette 2, the promoter for initiating transcription of the gene encoding the Nigri recombinase is Ptet, and the termination sequence is TrrnB; and / or, the replication initiation site is RK2s, wherein RK2s is a temperature-sensitive RK2 replication initiation site.

47. The complete set of products according to claim 46, characterized in that: The circular vector 3 is composed of the following elements in the order from 5' to 3': ParaBAD, the gene encoding Cre recombinase, TrrnB, Ptet, the gene encoding Nigri recombinase, TrrnB, the resistance gene, and RK2s.

48. The complete set of products according to claim 46 or 47, characterized in that: The nucleotide sequence of ParaBAD is SEQ ID NO:21; and / or The nucleotide sequence of the gene encoding the Cre recombinase is SEQ ID NO:22; and / or The nucleotide sequence of the TrrnB is SEQ ID NO:23; and / or The nucleotide sequence of the Ptet is SEQ ID NO:24; and / or The nucleotide sequence of the gene encoding the Nigri recombinase is SEQ ID NO:25; and / or The nucleotide sequence of the RK2s is SEQ ID NO:

26.

49. The complete set of products according to claim 42, characterized in that: The pinpoint input tool consists of the following 1)-3): 1) A targeting vector containing a coding sequence of sgRNA targeting the integration site in the genome of the host cell; 2) A homologous recombination fragment containing an upstream homologous arm, the specific DNA fragment, and a downstream homologous arm from the 5' to the 3' end; the upstream homologous arm is a fragment located upstream of the integration site in the genome of the host cell; the downstream homologous arm is a fragment located downstream of the integration site in the genome of the host cell; 3) An expression vector for expressing Cas protein and λ-red element.

50. The complete set of products according to claim 49, characterized in that: The targeted knock-in tool is composed of 1) and 2); and the ring carrier 3 also contains an expression cassette 3 for expressing the Cas protein and an expression cassette 4 for expressing the λ-red element.

51. The complete set of products according to claim 50, characterized in that: In expression cassette 3, the promoter for initiating transcription of the gene encoding the Cas protein is Psal, and the termination sequence is TrrnB; and / or, in expression cassette 4, the promoter for initiating transcription of the coding sequence of the λ-red element is Psal.

52. The complete set of products according to claim 51, characterized in that: The circular vector 3 is composed of the following elements in the order from 5' to 3': Psal, the gene encoding the Cas protein, TrrnB, Psal, exo-bet-gam gene, TλtL3 coding sequence, ColRNA coding sequence, ParaBAD, the gene encoding the Cre recombinase, TrrnB, Ptet, the gene encoding the Nigri recombinase, TrrnB, resistance gene, and RK2s.

53. The complete set of products according to any one of claims 42-52, characterized in that: The host cell can be a bacterium, fungus, animal cell, or plant cell.

54. The complete set of products according to claim 53, characterized in that: The host cell is Escherichia coli.

55. A method for multi-target, multi-fragment integration of the host cell genome, implemented using the kit product 5 described in any one of claims 42-54; comprising the following steps: (G1) Using a plurality of the aforementioned site-specific knock-in tools, the specific DNA fragment is knocked into a plurality of the aforementioned integration sites in the genome of the host cell; one specific DNA fragment is knocked into each of the aforementioned integration sites, and the specific DNA fragment knocked into different of the aforementioned integration sites is different; (G2) Connect the adapter sequences to both ends of several DNA fragments to be integrated, i.e., connect one end to P1 and the other end to P2; use homologous recombination to clone several DNA fragments to be integrated into several circular vectors 1 or several circular vectors 2 between P1 and P2 to obtain several recombinant vectors. (G3) The circular carrier 3 is introduced into the host cells treated with (G1); (G4) The recombinant vectors obtained in (G2) are introduced into the host cells treated in (G3), and after culturing and screening, recombinant cells with one copy of the DNA fragment to be integrated knocked into each of the several sites to be integrated are obtained. The number of items is 2-7.

56. A method for continuous multi-target, multi-fragment integration of the host cell genome, implemented using the kit product 6 described in any one of claims 42-54; the method comprising the following steps: (H1) The specific DNA fragment is knocked into the genome of the host cell at N sites to be integrated using N site-specific knock-in tools; one specific DNA fragment is knocked into each site to be integrated, and the specific DNA fragment knocked into different sites to be integrated is different; (H2) Connector sequences are attached to both ends of all the DNA fragments to be integrated, i.e., one end is connected to P1 and the other end is connected to P2; the DNA fragments to be integrated are grouped into groups of N, for a total of M groups, and the groups are numbered. Using homologous recombination, the N DNA fragments to be integrated from the odd-numbered groups are cloned between P1 and P2 in N circular vectors 1, respectively, to obtain several groups of recombinant vectors 1, each group containing N recombinant vectors 1, and each recombinant vector 1 carrying one DNA fragment to be integrated; using homologous recombination, the N DNA fragments to be integrated from the even-numbered groups are cloned between P1 and P2 in N circular vectors 2, respectively, to obtain several groups of recombinant vectors 2, each group containing N recombinant vectors 2, and each recombinant vector 2 carrying one DNA fragment to be integrated; (H3) The circular carrier 3 is introduced into the host cells treated with (H1); (H4) Introduce any one set of recombinant vector 1 or any one set of recombinant vector 2 obtained in (H2) into the host cells treated in (H3). After culturing and screening, recombinant cells with one copy of the DNA fragment to be integrated from the first group knocked into each of the N integration sites are obtained and are denoted as recombinant cells 1. Then, introduce another set of recombinant vector 2 or another set of recombinant vector 1 obtained in (H2) into recombinant cells 1. After culturing and screening, recombinant cells with one copy of the DNA fragment to be integrated from the first group and one copy of the DNA fragment to be integrated from the second group knocked into each of the N integration sites are obtained and are denoted as recombinant cells 2. In this step, if the first introduction is recombinant vector 1, then the second introduction is recombinant vector 2; if the first introduction is recombinant vector 2, then the second introduction is recombinant vector 1. (H5) Referring to (H4), in the alternating order of introducing the recombinant vector 1 and the recombinant vector 2, the recombinant vector 1 of all other groups and the recombinant vector 2 of all other groups are sequentially introduced into the recombinant cells obtained in the previous step. The final recombinant cells are recombinant cells in which one copy of the DNA fragment to be integrated from all groups is knocked into N integration sites at the same time. N is a natural number between 2 and 7; M is a natural number greater than or equal to 2.

57. The use of the complete set of DNA molecules of claim 40, or the DNA fragment or plasmid of claim 41, or the complete set of products of any of claims 42-54, in any of the following: (I1) Multi-target, multi-fragment integration of the host cell genome; (I2) Continuous multi-target and multi-fragment integration of the host cell genome.

58. A DNA molecule is any of the following: (J1) A DNA molecule with the nucleotide sequence SEQ ID NO:5 is denoted as nox4; (J2) A DNA molecule with the nucleotide sequence SEQ ID NO:6 is denoted as nox5; (J3) A DNA molecule with the nucleotide sequence SEQ ID NO:9 is denoted as nox8; (J4) A DNA molecule with the nucleotide sequence SEQ ID NO:10 is denoted as nox9; (J5) A DNA molecule with the nucleotide sequence SEQ ID NO:11 is denoted as nox10; (J6) A DNA molecule with the nucleotide sequence SEQ ID NO:12 is denoted as nox11.

59. A DNA fragment or plasmid containing the DNA molecule of claim 58.

60. The use of the DNA molecule of claim 58 or the DNA fragment or plasmid of claim 59 in the integration or sequential integration of the genome of a host cell.

61. A method for integrating exogenous DNA fragments into the genome of a eukaryotic cell, comprising the following steps: (K1) Knocking in a specific DNA fragment at an integration site in the genome of a eukaryotic cell; the specific DNA fragment contains a knock-in site-specific recombination site. (K2) expresses a knock-in site-specific recombinase in the eukaryotic cells described in (K1); (K3) Prepare circular DNA molecules containing the targeting fragment; The target fragment includes, in order from 5' to 3', the knock-in site-specific recombination site consistent with (K1) and the DNA fragment to be integrated; The knock-in site-specific recombination sites are nox, nox4, nox5, nox8, nox9, nox10, or nox11; The knock-in site-specific recombinase is Nigri recombinase; (K4) The circular DNA molecule obtained in (K3) is introduced into the eukaryotic cell described in (K2), and after culturing and screening, a copy of the DNA fragment to be integrated is knocked into the integration site in the genome of the eukaryotic cell.

62. The method according to claim 61, characterized in that: In the (K1) embodiment, the specific DNA fragment further includes a knockout site-specific recombination site I, which is located at the 5' end of the knock-in site-specific recombination site; In the (K2) embodiment, a knockout site-specific recombinase is also simultaneously expressed in the eukaryotic cells; In (K3), the targeting fragment includes, in order from 5' to 3', the knock-in site-specific recombination site II, the knock-out site-specific recombination site II, and the DNA fragment to be integrated, which are consistent with those in (K1). The knockout site-specific recombination site I and the knockout site-specific recombination site II are lox66 and lox71, respectively; or, the knockout site-specific recombination site I and the knockout site-specific recombination site II are loxF1 and loxF2, respectively. The knockout site-specific recombinase is Cre recombinase.

63. A method for the sequential integration of exogenous DNA fragments into the genome of a eukaryotic cell, comprising the following steps: (L1) A specific DNA fragment is knocked into the integration site in the genome of a eukaryotic cell. The specific DNA fragment consists of a knockout site-specific recombination site A and a knock-in site-specific recombination site from the 5' end to the 3' end. (L2) expresses knock-in site-specific recombinase and knock-out site-specific recombinase in the eukaryotic cells described in (L1); (L3) Prepare several circular DNA molecules; each circular DNA molecule contains a targeting fragment, and each targeting fragment contains a DNA fragment to be integrated; the several circular DNA molecules are divided into the following two categories according to the structural differences of the targeting fragments they contain: Category 1: Several circular DNA molecules 1 carrying a type I structure targeting fragment; the type I structure targeting fragment includes, in order from 5' to 3', the knock-in site specific recombination site B, the DNA fragment to be integrated, and the knock-out site specific recombination site C, which are consistent with those in (L1). Category 2: Several circular DNA molecules 2 carrying a type II structure targeting fragment; the type II structure targeting fragment includes, in order from 5' to 3', the knock-in site specific recombination site D, the DNA fragment to be integrated, and the knock-out site specific recombination site A, which is consistent with (L1); The knockout site-specific recombination site A, knockout site-specific recombination site B, knockout site-specific recombination site C, and knockout site-specific recombination site D are lox66, lox71, loxF1, and loxF2, respectively; or, the knockout site-specific recombination site A, knockout site-specific recombination site B, knockout site-specific recombination site C, and knockout site-specific recombination site D are loxF1, loxF2, lox66, and lox71, respectively; The knock-in site-specific recombination sites are nox, nox4, nox5, nox8, nox9, nox10, or nox11; The knock-in site-specific recombinase is Nigri recombinase; the knock-out site-specific recombinase is Cre recombinase; (L4) Introduce any one of the circular DNA molecules 1 obtained in (L3) into the eukaryotic cells of (L2), and after culturing and screening, obtain a recombinant cell with one copy of the first DNA fragment to be integrated knocked into the integration site, denoted as recombinant cell 1; then introduce the other circular DNA molecule 2 obtained in (L3) into the recombinant cell 1, and after culturing and screening, obtain a recombinant cell with one copy of the first DNA fragment to be integrated and one copy of the second DNA fragment to be integrated knocked into the integration site, denoted as recombinant cell 2; (L5) Referring to (L4), in the alternating order of introducing the circular DNA molecule 1 and the circular DNA molecule 2, all the remaining circular DNA molecules 1 and all the remaining circular DNA molecules 2 are sequentially introduced into the recombinant cell obtained in the previous step, that is, to achieve the insertion of one copy of all the DNA fragments to be integrated into the site to be integrated.

64. A method for library integration of eukaryotic cell genomes, characterized in that: The method includes steps (K1)-(K4) of claim 1, wherein the DNA fragment to be integrated is a library sequence; The library sequence is a collection of DNA fragments containing multiple independent target sequences, and each independent target sequence is integrated into the genome of a eukaryotic cell through steps (K1)-(K4) to obtain multiple integrated cell lines containing different target sequences.

65. A method for multi-site integration of the genome of a eukaryotic cell, comprising the following steps: (M1) Multiple different integration sites are selected in the genome of a eukaryotic cell, and a specific DNA fragment is knocked into each integration site; among them, The specific DNA fragments knocked into different integration sites are all different; The specific DNA fragment consists of a knockout site-specific recombination site I and a knock-in site-specific recombination site from the 5' end to the 3' end; (M2) expresses knock-in site-specific recombinase and knock-out site-specific recombinase in the eukaryotic cells of (M1); (M3) Prepare circular DNA molecules containing the targeting fragment for each integration site to obtain several circular DNA molecules; the targeting fragment includes the knock-in site-specific recombination site, the knock-out site-specific recombination site II, and the DNA fragment to be integrated in the order from 5' to 3'; wherein, for each integration site, the knock-in site-specific recombination site in the targeting fragment is the same as the knock-in site-specific recombination site knocked into the site in step (M1); The knockout site-specific recombination site I and the knockout site-specific recombination site II are lox66 and lox71, respectively; or, the knockout site-specific recombination site I and the knockout site-specific recombination site II are loxF1 and loxF2, respectively. The knock-in site-specific recombination site is nox, nox4, nox5, nox8, nox9, nox10 or nox11, and the knock-in site-specific recombination sites in any two different specific DNA fragments in (M1) are different from each other; The knock-in site-specific recombinase is Nigri recombinase; the knock-out site-specific recombinase is Cre recombinase; (M4) Several circular DNA molecules obtained in (M3) are introduced into the eukaryotic cell of (M2), and after culturing and screening, the DNA fragments to be integrated are knocked into different integration sites in the genome of the eukaryotic cell.

66. A complete product kit for integrating exogenous DNA fragments into the genome of eukaryotic cells, comprising: (f1) Fixed-point typing tool; The site-specific knock-in tool is capable of knocking in the specific DNA fragment described in step (K1) of claim 61 or 62 into the integration site in the genome of a eukaryotic cell; (f2) A recombinase or a nucleic acid molecule capable of expressing the recombinase or an expression cassette or recombinant plasmid containing the nucleic acid molecule; the recombinase is Nigri recombinase; or the recombinase is a combination of Nigri recombinase and Cre recombinase; (f3) Specific vector; the specific vector is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule described in step (K3) of claim 61 or 62.

67. A complete product for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells, comprising: (g1) A site-specific knock-in tool; the site-specific knock-in tool is capable of knocking in the specific DNA fragment described in step (L1) of claim 63 into the integration site in the genome of a eukaryotic cell; (g2) Nigri recombinase and Cre recombinase; or a nucleic acid molecule capable of expressing Nigri recombinase and Cre recombinase or an expression cassette or recombinant plasmid containing said nucleic acid molecule; (g3) Specific vector 1 and specific vector 2; the specific vector 1 is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule 1 described in step (L3) of claim 63; the specific vector 2 is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule 2 described in step (L3) of claim 63.

68. A complete product for multi-site integration of the genome of eukaryotic cells, comprising: (h1) A pinpoint typing tool; the pinpoint typing tool is capable of performing step (M1) of claim 65; (h2) Nigri recombinase and Cre recombinase; or a nucleic acid molecule capable of expressing Nigri recombinase and Cre recombinase or an expression cassette or recombinant plasmid containing said nucleic acid molecule; (h3) Several specific vectors; the several specific vectors are obtained by deleting only the DNA fragment to be integrated from the several circular DNA molecules described in step (M3) of claim 65.

69. The use of the complete product according to any one of claims 66-68 in any of the following: P1. Editing animal cell chromosomes; P2. Editing plant cell chromosomes; P3. Editing fungal cell chromosomes.

70. The use of the complete product according to any one of claims 66-68 in any of the following: Q1. Modification of eukaryotic microbial engineered strains; Q2. Construction of stable mammalian cell lines; Q3. Crop plant modification.

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