A method for serial editing of multiple sites of a chromosome in a eukaryotic cell

Abstract

The application discloses a method for continuous editing of multi-site chromosomes in eukaryotic cells. The application provides a method for integrating the genome of eukaryotic cells, comprising: using a knock-in site-specific recombination site (such as noxM) in the genome of eukaryotic cells, realizing integration of a large fragment circular DNA molecule at a specific site of the genome under the action of a knock-in site-specific recombinase (such as Nigri), and simultaneously using an adjacent knock-out site-specific recombination site (such as loxP or a mutant thereof), rearranging the related sites under the action of a knock-out site-specific recombinase (such as Cre), so as to realize continuity of integration. The novel editing platform of high-efficiency site-specific recombination provided by the application can realize directional, continuous and multi-site integration of a large fragment DNA in eukaryotic cells, and the application has important application prospects in the field of biological technologies including plant breeding, animal cell line development, fungus engineering and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for continuous editing of chromosomes at multiple sites in eukaryotic cells. Background Technology

[0002] In biotechnology research, constructing highly efficient engineered strains typically relies on multiple rounds of genetic optimization to progressively improve cell performance. This requires genome editing methods to possess high efficiency, large fragment carrying capacity, site integration flexibility, high integration efficiency, rapid reprogramming capabilities, and simplified DNA manipulation procedures. An ideal editing tool should be able to achieve stable, precise, and continuously operable large-fragment DNA integration in various eukaryotic systems.

[0003] Currently used gene editing technologies mainly include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system. These technologies largely rely on sequence-specific nucleases to introduce double-strand breaks (DSBs) into DNA at target sites, thereby achieving gene knock-in or knock-out through homologous directed repair (HDR) or non-homologous end joining (NHEJ) mechanisms. Although the CRISPR system exhibits high editing efficiency in eukaryotic cells, it still has limitations in integrating large DNA fragments and performing continuous multi-round editing, especially when integrating gene clusters exceeding several kb in length, where efficiency drops significantly.

[0004] Furthermore, HDR efficiency is low in most eukaryotic systems, particularly in non-dividing cells or difficult-to-transfect cell types (such as neurons, stem cells, and some protists). Although the efficiency can be improved to some extent by introducing exogenous recombinases or optimizing delivery systems, it is still difficult to meet the large-capacity, multi-site integration requirements for the construction of complex metabolic pathways or the integration of multi-gene circuits. Currently, editing strategies widely used in prokaryotes, such as the λ-Red and RecET homologous recombination systems, cannot be implemented in eukaryotes. CRISPR-based gene editing can be combined with transposon systems and leader editing systems, but it suffers from low efficiency, off-target effects, and the inability to achieve precise and continuous integration. In recent years, researchers have focused on improving editing precision, developing single-base editors, and expanding host applicability; however, achieving efficient and continuous integration of large DNA fragments in eukaryotic systems remains a key area that has not yet been fully explored. In multiple application scenarios such as metabolic engineering, synthetic biology, and gene therapy—e.g., plant trait improvement, construction of stable animal cell lines, and pathway optimization in eukaryotic microorganisms—there is an urgent need for genome editing tools that can support multi-site, long-fragment, continuous operation, and do not require complex DNA reconstruction.

[0005] Site-specific recombinases (SSRs) include tyrosine-based SSRs and serine-based SSRs. Tyrosine-based SSRs, such as Cre and Flp, are widely used in gene regulation and element integration in eukaryotic systems because they can recognize short-specific sites (such as loxP and FRT) and complete efficient recombination without cofactors. However, traditional SSR systems lack the ability to control the direction of integration, making it difficult to achieve efficient integration of large, continuous fragments, thus limiting their application in complex genome reconstruction. Serine-based SSRs can mediate recombination between attP and attB sites, forming attL and attR sites. However, their recognition sites are relatively long, and the protein molecules of these integrases are large, making their delivery into cells difficult. Furthermore, once attL and attR sites are formed, the system cannot be repeatedly recognized by the same integrase, making continuous gene editing difficult and limiting its potential application in multi-round editing. Therefore, developing a novel editing platform based on efficient SSR, enabling targeted, continuous, multi-site large-fragment DNA integration in eukaryotic cells, will have significant scientific value and broad application prospects, including in numerous biotechnology fields such as plant breeding, animal cell line development, and engineering modification of fungi and algae. Summary of the Invention

[0006] The purpose of this invention is to provide a method for continuous editing of chromosomes at multiple sites in eukaryotic cells.

[0007] In a first aspect, the present invention claims a method for integrating the genome of a eukaryotic cell.

[0008] The method for integrating exogenous DNA fragments into the genome of eukaryotic cells, as claimed in this invention, may include the following steps:

[0009] (A1) 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).

[0010] (A2) Expression of knock-in site-specific recombinase (hereinafter referred to as knock-in SSR) in the eukaryotic cells described in (A1); (A3) 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 as described in (A1); The knock-in SSRS can be nox, nox4, nox5, nox8, nox9, nox10 or nox11; The knock-in SSRS can be a Nigri recombinase; (A4) The circular DNA molecule obtained in (A3) is introduced into the eukaryotic cell described in (A2), 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.

[0011] Furthermore, in (A1), 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.

[0012] Furthermore, in (A2), a knockout site-specific recombinase (hereinafter referred to as knockout SSR) is also expressed simultaneously in the eukaryotic cells.

[0013] Furthermore, in (A3), 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, consistent with those in (A1).

[0014] 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.

[0015] Furthermore, the knocked-out SSR is a Cre recombinase.

[0016] The integration can be either insertion or replacement.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Alternatively, the upstream of the knockout SSRS-II segment may also contain a prokaryotic replication origin (such as ColE).

[0021] 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.

[0022] 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.

[0023] Furthermore, in step (A1), 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.

[0024] 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 (A1) 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 homologous arm and the downstream homologous arm are sequences located upstream and downstream of the integration site in the eukaryotic cell genome, respectively.

[0025] In other embodiments of the present invention, the site-specific knock-in tool may be a primeediting tool, i.e., a CRISPR-Cas nickase / reverse transcriptase-mediated technique to complete the step (A1). 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, an RNA molecule or expression vector expressing a Cas nickase (such as Cas9 nickase (H840A)) and a reverse transcriptase.

[0026] 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.

[0027] Further, in step (A2), the expression of Nigri recombinase and Cre recombinase in the eukaryotic cells of (A1) 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 into a vector while Cre recombinase is located within the circular DNA molecule.

[0028] Secondly, the present invention claims a method for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells.

[0029] 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: (B1) 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; (B2) Expression of knock-in SSR and knock-out SSR in the eukaryotic cells after treatment with (B1); (B3) 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 SSRS, knock-out SSRS-B, DNA fragment to be integrated, and knock-out SSRS-C, consistent with (B1); 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, consistent with (B1); 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. The knock-in SSRS can be nox, nox4, nox5, nox8, nox9, nox10 or nox11; The knock-in SSR can be Nigri recombinase; the knock-out SSR can be Cre recombinase. (B4) Introduce any one of the circular DNA molecules 1 obtained in (B3) into the eukaryotic cell of (B2), 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 (B3) 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; (B5) Referring to (B4), 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 or the selective insertion of the DNA fragments of interest to be integrated at the site to be integrated.

[0030] The integration can be either insertion or replacement.

[0031] 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.

[0032] 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.

[0033] 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).

[0034] 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.

[0035] 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.

[0036] Furthermore, in step (B1), 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.

[0037] In some embodiments of the present invention, the site-directed knock-in tool may be a CRISPR-Cas gene editing tool, i.e., step (B1) 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.

[0038] In other embodiments of the present invention, the site-specific knock-in tool may be a primeediting tool, i.e., a step (B1) mediated by Cas nickase / reverse transcriptase. 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 reverse transcriptase.

[0039] Further, in step (B2), the expression of Nigri recombinase and Cre recombinase in the eukaryotic cells of (B1) 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.

[0040] Thirdly, the present invention claims a method for library integration of the genome of eukaryotic cells.

[0041] The method for integrating a library of a eukaryotic cell genome claimed in this invention includes steps (A1)-(A4) of the first aspect above, 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 (A1)-(A4) to obtain multiple integrated cell lines containing different target sequences.

[0042] Fourthly, the present invention claims a method for multi-site integration of the genome of a eukaryotic cell.

[0043] The method for multi-site integration of the genome of a eukaryotic cell, as claimed in this invention, may include the following steps: (C1) 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 fragment knocked in at different integration sites is different from each other; The specific DNA fragment consists of knockout SSRS-I and knock-in SSRS from the 5' end to the 3' end; (C2) expresses knock-in SSR and knock-out SSR in the eukaryotic cells described in (C1); (C3) Prepare circular DNA molecules containing the targeting fragment for each integration site to obtain a number of 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 (C1); 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. 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 (C1) are different from each other; The knock-in SSR can be Nigri recombinase; the knock-out SSR can be Cre recombinase. (C4) Several circular DNA molecules obtained in (C3) are introduced into the eukaryotic cell of (C2), and after culturing and screening, the DNA fragments to be integrated are knocked into different integration sites in the genome of the eukaryotic cell.

[0044] In this method, the DNA fragments to be integrated can be different or the same, knocked into different sites.

[0045] The integration can be either insertion or replacement.

[0046] 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.

[0047] 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.

[0048] Furthermore, in step (C1), knocking in a specific DNA fragment to each of the sites to be integrated can be achieved using a site-specific knock-in tool.

[0049] In some embodiments of the present invention, the site-directed knock-in tool is a CRISPR-Cas gene editing tool, that is, step (C1) 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.

[0050] Further, in step (C2), the expression of Nigri recombinase and Cre recombinase in the eukaryotic cells treated in (C1) 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.

[0051] In this invention, the multiple sites can be 2-7 sites. In some embodiments of this invention, the multiple sites are 3 sites.

[0052] Fifthly, the present invention claims a kit for integrating exogenous DNA fragments into the genome of eukaryotic cells.

[0053] The kit product claimed in this invention for integrating exogenous DNA fragments into the genome of eukaryotic cells includes at least Nigri recombinase or a polypeptide having Nigri recombinase activity.

[0054] 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.

[0055] The complete product set also includes recombinase Cre or polypeptides with recombinase Cre activity.

[0056] 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.

[0057] Optionally, the kit may further include a protein with CAS activity, which can perform substrate recognition on specific sequences of eukaryotic chromosomes under the mediation of sgRNA. More selectively, proteins with CAS activity include Cas9, Ca12a, dCas9, etc. More selectively, proteins with CAS activity can be mutated to retain only nickase activity and fused with sequences possessing 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 mutants, loxP or its mutants, and combinations thereof, at specific locations on eukaryotic chromosomes. The aforementioned kit can efficiently integrate large fragments at any location on eukaryotic chromosomes. The integration site is determined by the pegRNA sequence.

[0058] The kit for integrating exogenous DNA fragments into the genome of eukaryotic cells claimed in this invention may include: (a1) A site-specific knock-in tool; the site-specific knock-in tool is capable of knocking in the specific DNA fragment described in step (A1) of the first aspect above into the integration site in the genome of a eukaryotic cell; (a2) 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; (a3) Specific vector; the specific vector is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule described in step (A3) of the first aspect above. The specific vector is used to insert the desired DNA fragment to be integrated to construct a new recombinant vector.

[0059] 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.

[0060] In other embodiments of the present invention, the site-specific knock-in tool may be a primeediting 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, an RNA molecule or expression vector expressing a Cas nickase (such as Cas9 nickase (H840A)) and a reverse transcriptase.

[0061] Sixthly, the present invention claims a kit for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells.

[0062] The kit product claimed in this invention for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells includes at least Nigri recombinase or a polypeptide having Nigri recombinase activity.

[0063] 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.

[0064] The complete product set also includes recombinase Cre or polypeptides with recombinase Cre activity.

[0065] 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.

[0066] Furthermore, the complete product 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; the complete product also includes a sequence capable of inserting lox(A)-noxM onto eukaryotic chromosomes.

[0067] 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.

[0068] 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.

[0069] Optionally, the kit may further include a protein with CAS activity, which can perform substrate recognition on specific sequences of eukaryotic chromosomes under the mediation of sgRNA. More selectively, proteins with CAS activity include Cas9, Ca12a, dCas9, etc. More selectively, proteins with CAS activity can be mutated to retain only nickase activity and fused with sequences possessing 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 mutants, loxP or its mutants, and combinations thereof, at specific locations on eukaryotic chromosomes. The aforementioned kit can efficiently integrate large fragments at any location on eukaryotic chromosomes. The integration site is determined by the pegRNA sequence.

[0070] The kit for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells, as claimed in this invention, may include: (b1) A site-specific knock-in tool; the site-specific knock-in tool is capable of knocking in the specific DNA fragment described in step (B1) of the second aspect above into the integration site in the genome of a eukaryotic cell; (b2) 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; (b3) 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 (B3) of the second aspect 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 (B3) of the second aspect 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.

[0071] Seventhly, the present invention claims a kit for multi-site integration of the genome of eukaryotic cells.

[0072] The kit for multi-site integration of the genome of eukaryotic cells claimed in this invention may include: (c1) Fixed-point typing tool; the fixed-point typing tool can perform the steps (C1) of the fourth aspect above. (c2) 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; (c3) 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 (C3) of the fourth aspect 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.

[0073] In the aforementioned relevant aspects, the recombinase Cre and its recognition sequence can be replaced by recombinases such as FLP, Dre, and Panta, and their recognition sequences.

[0074] In all the aforementioned relevant aspects, the DNA fragment to be integrated is a fragment of 60 kb or less.

[0075] In the aforementioned related aspects, the nucleotide sequence of nox is shown in SEQ ID NO:1; the nucleotide sequence of nox4 is shown in SEQ ID NO:2; the nucleotide sequence of nox5 is shown in SEQ ID NO:3; the nucleotide sequence of nox8 is shown in SEQ ID NO:4; the nucleotide sequence of nox9 is shown in SEQ ID NO:5; the nucleotide sequence of nox10 is shown in SEQ ID NO:6; and the nucleotide sequence of nox11 is shown in SEQ ID NO:7.

[0076] In the aforementioned related aspects, the nucleotide sequence of lox66 is shown in SEQ ID NO:8; the nucleotide sequence of lox71 is shown in SEQ ID NO:9; the nucleotide sequence of loxF1 is shown in SEQ ID NO:10; and the nucleotide sequence of loxF2 is shown in SEQ ID NO:11.

[0077] Eighthly, the present invention claims protection for the use of the complete set of products described in aspects five through seven above in any of the following: P1. Editing animal cell chromosomes; P2. Editing plant cell chromosomes; P3. Editing fungal cell chromosomes.

[0078] The fungal cells may include, but are not limited to: Yersinia lipolytica, Pichia pastoris, Hansenula polymorpha, Saccharomyces cerevisiae, Kluyveromyces genus, Trichoderma genus, and Aspergillus genus.

[0079] 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.

[0080] 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.

[0081] Ninthly, the present invention claims protection for the use of the complete set of products described in aspects five through seven above in any of the following: Q1. Modification of eukaryotic microbial engineered strains; Q2. Construction of stable mammalian cell lines; Q3. Crop plant modification.

[0082] 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.

[0083] 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.

[0084] In some embodiments of the present invention, the crop plant modification specifically refers to the modification of the stress resistance traits of corn plants.

[0085] This invention enables rapid and continuous genome integration using simple molecular biology operations, and can achieve large-fragment / continuous integration of eukaryotic genomes. In addition, it can also be used to mediate the integration process through CRISPR. Attached Figure Description

[0086] Figure 1 This diagram illustrates the process of integrating large, continuous gene segments into eukaryotic cells using this method.

[0087] Figure 2 Gene annotation based on sequencing results after integration of the MVA1 fragment at the POX5 position of the Yersinia lipophila genome in Example 5.

[0088] Figure 3 The results of multiple rounds of integration of the *Yarrowia lipophila* genome in Example 6 are shown. PN represents the number of positive clones; PR represents the positive integration rate. 1-3 represent the first, second, and third rounds of integration experiments, respectively.

[0089] Figure 4 Primers for gene editing detection in CHO cells in Examples 9 and 10 were designed. Among them, (A) is the design of primers for identifying the EF1α promoter library (protein expression library) in Example 9; (B) is the design of primers for identifying the CD19 mutant library in Example 10.

[0090] Figure 5 The positive integration efficiency of CHO cells in Examples 9 and 10 is given. "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). Detailed Implementation

[0091] 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.

[0092] 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.

[0093] In the following embodiments, this method is described as an "Ein-out method" (Genome). E diting through the combination of targeted knock- in and knock- out ). Figure 1 This diagram illustrates the process of integrating large, continuous gene segments into eukaryotic cells using this method.

[0094] The experimental procedures in the following examples have all been repeatedly verified, and the experimental results are all statistically significant. The specific sequences of each DNA fragment involved in the following examples are shown in Table 1, and the sequences of each primer involved in the following examples are shown in Table 2.

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] Example 1, General Experimental Method (1) Obtaining DNA fragments 1-1-a) PCR amplification method 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. PCR products were recovered using the EasyPure DNA Gel Purification Kit (TransGen Biotech, catalog number: EG101-01) to obtain DNA fragments.

[0119] (2) Obtaining plasmids 1-2-a) Total Synthesis Method Plasmids were obtained using a whole-genome synthesis method.

[0120] 1-2-b) Gibson splicing and assembly method 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.

[0121] 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.

[0122] 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.

[0123] 1-2-c) T4-DNA ligase ligation method 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.

[0124] 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).

[0125] 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. The plasmids were extracted from correctly identified positive clones to obtain positive plasmids.

[0126] Information on the DNA fragments involved in this invention is shown in Table 1, and information on the primers used is shown in Table 2.

[0127] Example 2: Transformation and screening of Yersinia lipophila (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 YeastTransformation II Kit, catalog number T2001, brand ZYMO RESEARCH.

[0128] (2) Screening of transformants 2-2-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.

[0129] The culture medium formula used is as follows: 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.

[0130] 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.

[0131] 2-2-b) Commonly used screening markers and their working concentrations Hygromycin B resistance marker: Its expression product, hygromycin B phosphotransferase, can induce resistance in cells to hygromycin B. Working concentration: 300-500 µg / mL.

[0132] Noristrine resistance marker: Its expression product, noristrine acetyltransferase, can induce resistance in cells to noristrine. Working concentration: 100-200 µg / mL.

[0133] 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.

[0134] 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.

[0135] Some examples of tags include: 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.

[0136] Example 3: Transformation and Screening of Mammalian Cells (1) Cell preparation 3-1-a) Preparation of CHO cells: 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 incubated at 37°C in a 5% CO2 incubator to achieve 70%-90% confluence at the time of transformation.

[0137] 3-1-b) Preparation of primary cells such as T cells: See subsequent examples for details, depending on the source of the cells.

[0138] (2) Conversion method Any one of these embodiments may be selected in subsequent implementations.

[0139] 3-2-a) Liposome conversion (using Lipofectamine 3000 as an example): It is suitable for 293T cells, CHO cells, and some easily transfected primary cells.

[0140] Step 1: Complex preparation In serum-free medium such as Opti-MEM, mix plasmid DNA with P3000™ enhancer (solution A). In another tube, mix Lipofectamine 3000 transfection reagent with Opti-MEM (solution B). After incubating at room temperature for 5 minutes, mix solutions A and B and let stand at room temperature for 15-20 minutes to form a DNA-liposome complex.

[0141] Step 2: Conversion 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.

[0142] Step 3: Cultivation 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.

[0143] 3-2-b) Electroconversion method: Suitable for CHO cells, difficult-to-transfect primary cells (such as T cells and neuronal stem cells), and suspension culture of 293T cells.

[0144] Step 1: Preparation of cell suspension Digest adherent cells with trypsin or resuspend the cells in buffer and collect them by centrifugation. Wash the cells thoroughly 1-2 times with pre-cooled 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.

[0145] Step 2: Electric shock 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 250-350 V, capacitance 950 μF; for primary T cells: voltage 1300-1500 V, pulse width 10-30 ms). Perform electroporation.

[0146] Step 3: Recovery 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.

[0147] (3) Screening of transformants 3-3-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.

[0148] 3-3-b) Commonly used screening markers and their working concentrations Puromycin resistance marker: Its expression product, puromycin-N-acetyltransferase, can induce cell resistance to puromycin.

[0149] 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).

[0150] G418 (Genzyme) resistance marker: Its expression product, neomycin phosphotransferase, can make cells resistant to G418.

[0151] 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).

[0152] Hygromycin B resistance marker: Its expression product, hygromycin B phosphotransferase, can induce cell resistance to hygromycin B.

[0153] Working concentration: 50-400 µg / mL (e.g., 100-200 µg / mL is commonly used for mammalian cells).

[0154] Example 4: Transformation and Screening of Plant Cells (1) Preparation of the receptor system 4-1-a) Induction and culture of callus tissue: 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 under low light for 2-4 weeks to induce the formation of loose, pale yellow embryogenic callus.

[0155] 4-1-b) Establishment of suspension cell lines: The embryogenic callus tissue was transferred to liquid culture medium and cultured with shaking 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.

[0156] 4-1-c) Preparation of the bladed disc: 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.

[0157] (2) Conversion method 4-2-a) Agrobacterium-mediated transformation: It is suitable for most dicotyledonous and monocotyledonous plants (such as rice, tobacco, and Arabidopsis thaliana).

[0158] Step 1: Agrobacterium culture 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.

[0159] Step 2: Co-culture 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.

[0160] Step 3: Sterilization and Screening 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.

[0161] 4-2-b) Gene gun transformation: It is suitable for plants that are not easily infected by Agrobacterium, especially important cereal crops (such as corn and wheat).

[0162] Step 1: Micro-elastic wrapping Plasmid DNA is encapsulated on the surface of a microelastic carrier such as gold powder or tungsten powder.

[0163] Step 2: Bombardment 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.

[0164] Step 3: Recovery and Filtering 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.

[0165] (3) Screening and regeneration of transformants 4-3-a) Screening for resistant callus: 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.

[0166] 4-3-b) Plant regeneration: 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.

[0167] 4-3-c) Commonly used screening markers and their working concentrations Hygromycin B resistance marker: Its expression product, hygromycin B phosphotransferase, can induce plant cells to develop resistance to hygromycin B.

[0168] Working concentration: 10-50 mg / L (for use in culture media).

[0169] Kanamycin / Genomycin resistance markers: their expression product, neomycin phosphotransferase, can induce resistance in plant cells to kanamycin or G418.

[0170] Working concentration: 50-100 mg / L (for use in culture media).

[0171] Glufosinate resistance marker: Its expression product, phosphinic acid acetyltransferase, can induce plant cells to develop resistance to glufosinate.

[0172] Working concentration: 2-10 mg / L (for use in culture media).

[0173] Example 5: Chromosome editing of Yersinia lipophila strain using the Ein-out method The plasmids and DNA fragments were prepared in accordance with Example 1.

[0174] The transformation and screening of Yeast lipophila were carried out according to Example 2.

[0175] (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: 5-1-a) Targeting a specific coding region of the POX5 gene, the following sgRNA target sequence was designed: 5'-GCTGGGTCACGGATCTAACG-3'. 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 1 for the sequence of each element).

[0176] 5-1-b) The following DNA fragment P1LN was artificially synthesized: 5'-POX5up-lox66-nox-POX5down-3' (the sequence of this 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 1). Using this fragment as a template and P-1 / P-2 as primers (sequences are shown in Table 2), homologous recombination fragments were prepared by PCR amplification.

[0177] 5-1-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 2). 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. This resulted in strain P01, which integrates the lox66-nox sequence at the POX5 site on the genome.

[0178] (2) Integrating the MVA1 fragment into Yersinia lipophila using the “Ein-out” method 5-2-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 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 1). 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.

[0179] 5-2-b) pNL-MVA1 plasmid transformation. Containing the MVA1 fragment (P... TEF1 -ERG10-T cyc1 -P TEF1 -ERG13-T cyc1 -P TEF1 -HMG2-T cyc1 -P TEF1 -ERG12-T cyc1The 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 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 1). 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.

[0180] 5-2-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 2). 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.

[0181] 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.

[0182] 5-2-d) Deletion of HygR1 selection marker: The pCre-Y1 plasmid was introduced via chemical transformation. The pCre-Y1 plasmid was entirely synthetic, and its structural composition is as follows: 5'-P TEF1 -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 1). 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 2). 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.

[0183] 5-2-e) To verify the efficiency of Nigri-mediated integration, the following control was set up in processes 5-2-a) to 5-2-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.

[0184] Results analysis (Table 3): In experiments 5-2-b) and 5-2-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 5-2-e), replacing pNigri-Y1 with pCre-Y1 resulted in no transformant growth on the plate. Sequencing of positive clones from 5-2-c) and 5-2-d) showed that none of the knock-in sequences in all positive clones were mutated, and only one nox site remained on the genome after the selection marker deletion, allowing for further precise integration. Figure 2 The above results demonstrate that the method provided by this invention can efficiently achieve editing in Yersinia lipophila.

[0185] Table 3. Efficiency of MVA1 fragment integration at the POX5 position in the Yersinia lipolyticis genome tool plasmids pNigri-Y1 pCre-Y1 Positive integration rate 100% - Number of positive clones (PN / μg DNA) <![CDATA[(1.23±0.10)×10 4 ]]> 0 Example 6: Sequential chromosome editing of *Yarrowia lipophila* strain using the Ein-out method. The plasmids and DNA fragments were prepared in accordance with Example 1.

[0186] The transformation and screening of Yeast lipophila were carried out according to Example 2.

[0187] (1) The first round of editing was completed in accordance with the process in Example 5. The strain was identified by the process described in Example 5. The positive clone with the HygR1 resistance gene eliminated was named P02.

[0188] (2) The second round of editing was completed exactly as described in Example 5, 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 synthesized entirely artificially, and its structure 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 1). 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 after transforming P02 was identified as described in Example 5, except that primer N-7 / MVA-2 (sequence shown in Table 2) 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.

[0189] (3) The third round of editing was completed exactly as described in Example 5, 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 synthesized entirely artificially, and its structure 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 1). 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 5, except that primer N-8 / MVA-3 (sequence shown in Table 2) 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.

[0190] Results analysis: 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. 4PN / μg DNA, (5.78±0.32)×10 3 PN / μg DNA, (1.01±0.09)×10 3 PN / g DNA ( Figure 3 The above results demonstrate that the method provided by this invention can achieve highly efficient continuous editing in Yersinia lipophila.

[0191] Example 7: Large-segment chromosome editing of *Yarrowia lipophila* strain using the Ein-out method. The plasmids and DNA fragments were prepared in accordance with Example 1.

[0192] The transformation and screening of Yeast lipophila were carried out according to Example 2.

[0193] The editing was completed exactly as described in Example 5, 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 1). 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 2) 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.

[0194] Results analysis: 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*.

[0195] Example 8: Multi-site editing of chromosomes in *Yarrowia lipophila* strains using the Ein-out method. The plasmids and DNA fragments were prepared in accordance with Example 1.

[0196] The transformation and screening of Yeast lipophila were carried out according to Example 2.

[0197] The editing was completed exactly as described in Example 5, except that: 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 5. The insertion of lox66-nox into the POX5 locus was exactly the same as in Example 5. 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 2) were used instead of P-1 / P-2; and primers N-3 / N-4 (sequences shown in Table 2) 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 1 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 1 for the sequence of each element). The operational differences when 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 2) are used instead of P-1 / P-2; during PCR verification, primers N-5 / N-6 (sequences shown in Table 2) are used instead of N-1 / N-2, and a positive result is achieved when a target band of approximately 2000 bp 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. The sequence of each element is shown in Table 1). 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. The sequence of each element is shown in Table 1).

[0198] The transformation process involved the simultaneous transformation of pNL-MVA1 plasmid (see Example 5), 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.

[0199] Results analysis: 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. 2PN / 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*.

[0200] Example 9: Constructing a protein expression library using CHO cells The plasmids and DNA fragments were prepared in accordance with Example 1.

[0201] The transformation and screening of CHO cells were carried out according to Example 2.

[0202] (1) Construction of CHO-H11-LN cell line 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: 9-1-a) Target design and sgRNA expression plasmid construction Designed sgRNA target sequence H11-sgRNA1: 5'-TATACACTTGAGCCAGTAGTGGG-3'.

[0203] 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.

[0204] Primers designed: H11-sgRNA1-F / H11-sgRNA1-R (sequences are shown in Table 2).

[0205] 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 1 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.

[0206] 9-1-b) Preparation of NL fragment targeting plasmid pH11-NL 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 1).

[0207] 9-1-c) Cell transfection and screening 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 2). A band of approximately 2300 bp was observed upon successful NL integration. Positive cell lines were named CHO-H11-LN.

[0208] (2) Constructing a CHO cell library containing an EF1α promoter library using EIN-OUT 9-2-a) The 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 1 for the sequence of each element).

[0209] 9-2-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 2) with an error-prone PCR kit (Shanghai Zeye Company, catalog number ZY6351PD).

[0210] The vector fragment was amplified by PCR using pNL1-PEF-GFP plasmid as a template and primers V-1 / V-2 (sequences are shown in Table 2).

[0211] Following the Gibson method in Example 1, the vector fragment and the mutant library fragment were ligated to obtain the plasmid library pLib-EF.

[0212] The 9-2-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 this plasmid is obtained by sequentially connecting the above elements from the 5' end to the 3' end. See Table 1 for the sequence of each element).

[0213] 9-2-d) Seed CHO-H11-LN cells into suitable multi-well plates or culture flasks, and transfect when the density reaches 70-80%. Use electroporation to co-introduce the pNigri-C1 plasmid and pLib-EF plasmid library into the cells. 48 hours after transfection, replace the medium with selection medium containing hygromycin (300 μg / mL) and doxycycline (1 μg / mL). Continue selection for 2-3 days, then replace the medium with selection medium containing hygromycin (300 μg / mL) and culture 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. Use flow cytometry to sort cells that only show green fluorescence signals but no red or blue fluorescence signals. The selected cells should be those whose fragments have been integrated into the chromosome, and whose redundant sequences (including one nox site after recombination) have been eliminated.

[0214] 9-4-c) Identification and screening of EF1α promoter libraries CHO cell lines containing the EF1α promoter library were cultured in 10 cm plates. A portion of the cells were harvested and screened using flow cytometry for cells with different green fluorescence signals. 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 identify the efficiency of Ein-out specific integration. During the identification process, multiple primer sets were used for PCR amplification to achieve accurate typing, such as... Figure 4 As shown in (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) Amplification was performed using primer pairs N-11 and N-18 to distinguish between integration-negative cells and heterozygous cells; (3) Amplification was performed using primers N-11 and N-12 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-13 / N-14, N-15 / N-17, and N-12 / N-16 to determine whether there were randomly inserted cells. The PCR identification results corresponding to cells of different genotypes are shown in Table 4. The mutated EF1α promoter sequence was obtained by PCR amplification using primers N-9 / N-10 and then sequencing. The sequences of the primers involved above are shown in Table 2.

[0215] Table 4. PCR identification results of cells with different genotypes Primer combination Integration positive (bp) Integration negative (bp) Heterozygous (bp) Incomplete integration (bp) Random integration (bp) N-11 / N-14 400 Unable to amplify / / Unable to amplify N-16 / N-18 2500 Unable to amplify / 2500 Unable to amplify N-11 / N-18 4000 1800 Multiple amplification bands 1800 or N-11 / N-12 Unable to amplify Unable to amplify / 2500 / N-13 / N-14 Unable to amplify Unable to amplify / 2900 2900 or N-15 / N-17 1900 Unable to amplify / / 1900 or N-12 / N-16 Unable to amplify Unable to amplify / / 3300 or 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 at the same time: (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".

[0216] The results are as follows Figure 5 As shown, the integration positive cell rate of the above CHO expression library (the ratio of integration positive cells identified by PCR to the total number of cells) is above 73.45±4.45%. The method provided by this invention has significant advantages in constructing expression libraries (EF1α promoter libraries) in CHO cells: (1) it minimizes the probability of non-specific integration such as random integration, and greatly improves the quality of the library; (2) after the library is constructed, it can be quickly quality controlled and screened using flow cytometry, shortening the working cycle; (3) the integration site retains a single nox site, which can be quickly used for the next round of gene editing without additional genome modification.

[0217] Example 10: Integration and screening of continuous chromosome libraries in CHO cells using the Ein-out method. The plasmids and DNA fragments were prepared in accordance with Example 1.

[0218] The transformation and screening of CHO cells were carried out according to Example 2.

[0219] (1) First round of library integration and screening based on EF1α promoter library The method is the process described in Example 9. In this process, the integrated fragment P is found in different cells sorted by flow cytometry. 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 PEF1α -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 9, 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 of elements linked sequentially, see Table 1 for the sequence of each element).

[0220] (2) Second round of library integration and screening based on CD19 mutant libraries The 10-2-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 1 for the sequence of each element).

[0221] 10-2-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 2) with an error-prone PCR kit (Shanghai Zeye Company, catalog number ZY6351PD).

[0222] The vector fragment was amplified by PCR using pNL2-CD19 plasmid as template and primers V-1 / V-3 (sequences are shown in Table 2).

[0223] Following the Gibson method in Example 1, the vector fragment and the mutant library fragment were ligated to obtain the plasmid library pLib-CD19.

[0224] 10-2-c) Seed CHO-EF1-WO3 cells into suitable multi-well plates or culture flasks, and transfect when the cell 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.

[0225] 10-2-d) Identification and screening of CD19 mutant libraries Cell banks containing the CD19 mutant library were cultured in 10 cm 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 identify the efficiency of Ein-out specific integration. Multiple primer sets were used for PCR amplification during the identification process to achieve accurate typing, such as... Figure 4 As shown in (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) Amplification was performed using primer pairs N-11 and N-18 to distinguish between integration-negative cells and heterozygous cells; (3) Amplification was performed using primers N-11 and N-12 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 randomly inserted cells were present. The PCR identification results for cells of different genotypes are shown in Table 5. The sequences of the primers involved are shown in Table 2.

[0226] Table 5. PCR identification results of cells with different genotypes Primer combination Integration positive (bp) Integration negative (bp) Heterozygous (bp) Incomplete integration (bp) Random integration (bp) N-11 / N-19 1500 Unable to amplify / / Unable to amplify N-18 / N-21 1350 Unable to amplify / 1350 Unable to amplify N-11 / N-18 4700 4000 Multiple amplification bands / 4000 or N-11 / N-12 Unable to amplify Unable to amplify / 4600 / N-16 / N-19 Unable to amplify Unable to amplify / 6200 / N-20 / N-22 1800 Unable to amplify / / 1800 or N-12 / N-21 Unable to amplify Unable to amplify / / 2000 or 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 at the same time: (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 to obtain a 4000bp product; primers N-20 / N-22 amplify to obtain an 1800bp product; primers N-12 / N-21 amplify to obtain a 2000bp product. If not met, it is classified as "unidentified".

[0227] The results showed that 98% of the sorted cells could amplify the specific integration target band (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%. Figure 5 As shown above, the method provided by this invention enables rapid, continuous, and highly efficient library integration onto the genome.

[0228] Example 11: Construction of a stable CAR-T expression cell line The plasmids and DNA fragments were prepared in accordance with Example 1.

[0229] The transformation and screening of T cells were carried out according to Example 2.

[0230] (1) Isolation and activation of peripheral blood T cells from patients 11-1-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.

[0231] 11-1-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.

[0232] 11-1-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 in a 5% CO2 incubator for approximately 48 hours to activate T cells.

[0233] (2) Establish a stable CAR expression cell line using the “Ein-out” method 11-2-a) Preparation of pegRNA For the CCR5 gene locus, pegRNAs based on twinPE leader editing were designed, and 2'-O-methyl modified pegRNAs, CNpegRNA1 and CNpegRNA2, were synthesized by Shanghai Sangon Biotech Co., Ltd. (sequences are shown in Table 1).

[0234] 11-2-b) Preparation of Cas9 nickase-reverse transcriptase fusion protein (PE2) mRNA The commercial plasmid pCMV-PE2 (addgene, #132775) was linearized using PmeI restriction endonuclease (NEB, catalog number R0560L), and then 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).

[0235] 11-2-c) Preparation of Nigri mRNA The pCMV-Nigri plasmid was synthesized entirely artificially. Specifically, it was obtained by replacing the Cas9 reading frame in the pCMV-PE2 plasmid with the Nigri gene (sequence shown in Table 1). The preparation method of Nigri mRNA was completely consistent with the method in 11-2-b), except that the pCMV-PE2 plasmid was replaced with the pCMV-Nigri plasmid.

[0236] 11-2-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 1 for the sequence of each element). CAR is the gene encoding the chimeric antibody used in CAR-T cell therapy for multiple myeloma.

[0237] Integration of 11-2-e) pNox-CAR: Take 5×10 5 Cells activated in step (1) were resuspended in B1mix electroporation buffer, and 2 μg of mRNA prepared in step 11-2-b), 2 μg of mRNA prepared in step 11-2-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 and the medium was changed. 72 hours after transfection, the cells were collected for genomic DNA extraction and subsequent analysis.

[0238] 11-2-f) Screening and gene integration analysis of positive cell lines 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 using Nanodrop or Qubit. The target genomic region was first amplified using forward and reverse primer sequences (see Table 2). 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 with 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.

[0239] Results analysis: In the 11-2-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.

[0240] Example 12: Gene insertion into rice protoplasts using the Ein-out method The plasmids and DNA fragments were prepared in accordance with Example 1.

[0241] The transformation and screening of T cells were carried out according to Example 2.

[0242] (1) Preparation of rice protoplasts Healthy 3-4 leaf stage rice seedlings of Zhonghua 1 were selected and enzymatically hydrolyzed at 28℃ in the dark with shaking for 4-5 hours 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.

[0243] (2) Utilizing Ein-out to integrate target genes 12-2-a) Preparation of pCP-RNA-Nigri plasmid 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 1). Among them, PpegRNA1 contains lox66, and PpegRNA2 contains nox.

[0244] 12-2-b) Preparation of the targeting vector pNox-GFP plasmid 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 -P Os_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 1 for the sequence of each element).

[0245] 12-2-c) Transfection and screening of integrative plasmids High-quality plasmids were prepared using the Wizard Plus Midipreps DNA purification system. 5 μg of each plasmid prepared in 12-2-a) and 12-2-b) were mixed and used to transform the protoplasts prepared in Example 12(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 11-2-f).

[0246] In process 12-2-c), the integration positive efficiency based on Illumina sequencing can reach 82.8 ± 10.2%. The method provided by this invention avoids the disadvantages of other integrases, such as low integration direction efficiency, large molecular weight of integrases, and lack of continuous editing capability of integration sites. It also has advantages such as flexible editing design. It has promising application prospects in plant genome integration.

[0247] Example 13: Integrating drought-resistant genes into maize plant chromosomes using the "Ein-out" method. The plasmids and DNA fragments were prepared in accordance with Example 1.

[0248] The conversion and screening of corn were carried out according to Example 2.

[0249] (1) Plasmid preparation 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 1). Among them, PpegRNA3 contains lox66 and PpegRNA4 contains nox.

[0250] 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 1 for the sequence of each element).

[0251] 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 1 for the sequence of each element).

[0252] 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 1 for the sequence of each element).

[0253] (2) Construction of the Z01 strain 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, with no other additional indel mutations. The successfully validated line was named Z01.

[0254] (3) Integration of large-segment stress-resistance genes Z01 callus tissue was spread evenly on hypertonic medium and pre-dried. The pM-Nigri and pNox-Zm plasmids prepared in Example 13(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.

[0255] 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.

[0256] 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.

Claims

1. A method for integrating exogenous DNA fragments into the genome of a eukaryotic cell, comprising the following steps: (A1) A specific DNA fragment is knocked into an integration site in the genome of a eukaryotic cell; the specific DNA fragment contains a knock-in site-specific recombination site; (A2) Expression of a knock-in site-specific recombinase in the eukaryotic cells described in (A1); (A3) Prepare a circular DNA molecule containing a targeting fragment; the targeting fragment includes, in order from 5' to 3', the knock-in site-specific recombination site and the DNA fragment to be integrated, consistent with those in (A1); The knock-in site-specific recombination site is nox, nox4, nox5, nox8, nox9, nox10, or nox11; The knock-in site-specific recombinase is Nigri recombinase; (A4) The circular DNA molecule obtained in (A3) is introduced into the eukaryotic cell described in (A2), 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.

2. The method according to claim 1, characterized in that: In (A1), 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 (A2), a knockout site-specific recombinase is also expressed simultaneously in the eukaryotic cells; In (A3), the target 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 (A1). 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.

3. A method for the sequential integration of exogenous DNA fragments into the genome of a eukaryotic cell, comprising the following steps: (B1) A specific DNA fragment is knocked into the integration site in the genome of a eukaryotic cell, the specific DNA fragment consisting of a knockout site-specific recombination site A and a knock-in site-specific recombination site from the 5' end to the 3' end; (B2) Expression of knock-in site-specific recombinase and knock-out site-specific recombinase in the eukaryotic cells described in (B1); (B3) 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 (B1). 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 (B1). 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 site is 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; (B4) Introduce any one of the circular DNA molecules 1 obtained in (B3) into the eukaryotic cell of (B2), 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 (B3) 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; (B5) Referring to (B4), 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.

4. A method for integrating a library of eukaryotic cell genomes, characterized in that: The method includes steps (A1)-(A4) 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 (A1)-(A4) to obtain multiple integrated cell lines containing different target sequences.

5. A method for multi-site integration of the genome of a eukaryotic cell, comprising the following steps: (C1) Select multiple different integration sites in the eukaryotic cell genome, and knock in a specific DNA fragment into each integration site; where, 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; (C2) expresses knock-in site-specific recombinase and knock-out site-specific recombinase in the eukaryotic cells described in (C1); (C3) Prepare circular DNA molecules containing the targeting fragment for each integration site to obtain several circular DNA molecules; the targeting fragment includes, in order from 5' to 3', the knock-in site-specific recombination site, the knock-out site-specific recombination site II, and the DNA fragment to be integrated; 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 at that site in step (C1); 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 (C1) are different from each other; The knock-in site-specific recombinase is Nigri recombinase; the knock-out site-specific recombinase is Cre recombinase; (C4) Several circular DNA molecules obtained in (C3) are introduced into the eukaryotic cell of (C2), and after culturing and screening, the DNA fragments to be integrated are knocked into different integration sites in the genome of the eukaryotic cell.

6. A complete product kit for integrating exogenous DNA fragments into the genome of eukaryotic cells, comprising: (a1) Fixed-point typing tool; The site-specific knock-in tool is capable of knocking in the specific DNA fragment described in step (A1) of claim 1 or 2 into the integration site in the genome of a eukaryotic cell; (a2) 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; (a3) Specific vector; the specific vector is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule described in step (A3) of claim 1 or 2.

7. A complete product for the continuous integration of exogenous DNA fragments into the genome of eukaryotic cells, comprising: (b1) Fixed-point typing tool; The site-specific knock-in tool is capable of knocking in the specific DNA fragment described in step (B1) of claim 3 into the integration site in the genome of a eukaryotic cell; (b2) 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; (b3) 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 (B3) of claim 3; the specific vector 2 is obtained by deleting only the DNA fragment to be integrated from the circular DNA molecule 2 described in step (B3) of claim 3.

8. A complete product for multi-site integration of the genome of eukaryotic cells, comprising: (c1) A pinpoint typing tool; the pinpoint typing tool is capable of performing step (C1) of claim 5. (c2) 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; (c3) 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 (C3) of claim 5.

9. The use of the complete set of products according to any one of claims 6-8 in any of the following: P1. Editing animal cell chromosomes; P2. Editing plant cell chromosomes; P3. Editing fungal cell chromosomes.

10. The use of the complete product according to any one of claims 6-8 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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