Cell Surface Tag Exchange (CSTE) System for Tracing and Manipulating Cells During Recombinase-Mediated Cassette Exchange Integration of Nucleic Acid Sequences into Engineered Receiver Cells

Abstract

To provide effective and improved methods for tracing and manipulation of engineered cells during an integration period.SOLUTION: The present invention provides a combined system comprising two separate components, where the first component is a tag-exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon, and a gene of interest (GOI) in the antisense orientation, and the second component is an engineered cell containing within its genome a tag-exchange receiver site (TERS), where recombinase mediated cassette exchange (RMCE) between the TEDV and TERS causes the derivative engineered cell to express the first CST and GOI, in place of the second CST and the reporter gene.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the fields of cell engineering and recombinant DNA technology. In particular, the present invention relates to systems and methods for creating, purifying, tracing and manipulating engineered cells during recombinase-mediated cassette exchange (RMCE)-based integration of a gene of interest. [Background technology]

[0002] Within the fields of genetic engineering and immunology, recombinase-mediated cassette exchange (RMCE) has become a useful tool for targeted genetic modification. See, for example, Turan et al., Gene (2013):515:1-27. Typically, post-RMCE methods for confirming the integration of a gene of interest (GOI) at the correct location require cell disruption and sample processing. A recently described approach overcomes this need for cell disruption and demonstrates that recombination events can be monitored and isolated by following the expression of a fluorescent marker protein (Phan et al., Sci Rep. (2017):7(1):17771). While the approach by Phan et al. avoids the need for cell disruption, it relies on intracellular protein fluorescence, making this technique unsuitable for high-content screening of integrated constructs due to the limited set of unique optical properties offered by fluorescent proteins. As a result, the number of genes that can be simultaneously labeled and detected using the described strategy is limited. Furthermore, intracellular fluorescent proteins are not amenable to physical manipulation to partition cells by substrate affinity methods such as magnetic activated cell sorting (MACS), which offers the potential for high throughput and parallelization in cell manipulation workflows or increased efficiency in the manipulation of high-content cell libraries.

[0003] While RMCE techniques currently exist, there is a need for tools for rapid and robust cell manipulation in both high-throughput and high-content applications, particularly when cells are co-transfected with pools of vectors containing pools of individual genes of interest, or when selection of cells by substrate affinity methods is desired. Thus, there is a clear need to provide effective and improved methods. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides a two-component cell surface tag exchange (CSTE) system for tracing and manipulating engineered cells during recombinase-mediated cassette exchange (RMCE)-based integration of a gene of interest (GOI). The first part of the CSTE system is a tag exchange donor vector (TEDV) encoding the GOI for integration along with a first cell surface tag (CST), where the first CST is flanked by a 3' fragment of intron sequence containing a splice acceptor site. This TEDV construct is flanked by RMCE elements. The second part of the CSTE is a tag exchange receiver site (TERS) contained within the genome of the engineered target cell, where the TERS encodes a selection gene and a second CST, where the second CST is flanked by a full-length intron sequence encoding an in-frame transmembrane domain, and the second RMCE element flanks the selection gene coding sequence. The CST encoded by TERS is essentially encoded as two exons connected by a single intron. The paired RMCE elements contained in TEDV and TERS are designed so that upon RMCE between the two constructs, the TEDV CST and GOI are exchanged in TERS, resulting in the loss of the TERS-encoded CST and selection gene. When unique CST exon sequences are exchanged by RMCE, the CST delivered by TEDV utilizes the TERS-encoded transmembrane domain. The promoter elements driving expression of CST and the selection gene / GOI from TERS are foreign to the RMCE-exchanged construct; therefore, the TEDV-delivered sequences are generally only expressed by faithful RMCE. This CSTE system allows rapid and reliable RMCE, conditionally confirmed by detection of affinity epitopes displayed by CST on the cell surface, and also allows physical partitioning of cells using substrate-immobilized affinity methods. This CSTE system also allows multiplexing of GOI integration and lineage tracing by using multiple affinity epitopes as CST elements. The CSTE system is a tool for rapid and robust cell engineering in both high-throughput and high-content applications.

[0005] In a first aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: A combination system comprising two separate components: a first component is a tag exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon flanked by 3' intron fragments and a gene of interest (GOI) in an antisense orientation; a second component is an engineered cell containing in its genome a tag exchange receiver site (TERS), the TERS encoding a second CST exon and encoding a reporter gene in an antisense orientation, the second CST exon being connected by a full-length intron sequence to an exon encoding a transmembrane domain; A combination system is provided in which paired recombinase-mediated cassette exchange (RMCE) elements are contained in TEDV and TERS, such that RMCE between TEDV and TERS results in the exchange of the TEDV-encoded GOI with the reporter element and the exchange of the second CST exon with the first CST exon, such that the derived engineered cells express the first CST and GOI instead of the second CST and reporter gene.

[0006] In one embodiment, the first component is a. First RMCE element - a 5' RMCE element encoded within a "non-functional" non-coding 3' intron fragment; b. a 3' intron fragment containing a branch point sequence, a polypyrimidine tract, and a 3' splice acceptor site; c. an exon containing CST encoded by TEDV in the 5'→3' direction; d. a first transcription terminator sequence for CST encoded in the 5' to 3' direction; e. a second transcription terminator for the GOI encoded 3'→5'; f. a sequence encoding the GOI in the 3' to 5' direction; g. Kozak sequence, h.5' RMCE element , wherein the CST exon and the first transcription terminator are encoded in the antisense direction from the GOI and associated transcription terminator and Kozak sequence.

[0007] In another embodiment, the second component is a. transcriptional promoter element; b. Kozak sequence, c. Type 2 membrane protein transmembrane domain exon, d. 5' intron splice donor site, e. a "non-functional" non-coding 3' intron fragment-encoded 5' RMCE element that is an equivalent of and pairs with the 5' RMCE element of TEDV; f. the functional sequence of the 3' intron fragment, including the branchpoint sequence, the polypyrimidine tract, and the 3' splice acceptor site; g. An exon containing a CST encoded by TERS in a 5' to 3' orientation (different from the CST encoded by TEDV); h. a transcription terminator sequence for CST encoded in the 5' to 3' direction; i. a transcription terminator sequence for the 3' to 5' sequence; j. a sequence encoding a selection gene in the 3' to 5' direction; k. Kozak sequence for efficient translation initiation of selected gene transcripts; l.3'RMCE element, 3' genomic elements responsible for regulating expression and tracking of m.CST transcripts wherein the transmembrane domain exon and the CST exon are encoded in the antisense direction from the reporter gene, and a first transcriptional promoter element drives transcription of the combination of the transmembrane domain and CST, and a second transcriptional promoter element drives transcription of the reporter gene.

[0008] In a second aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: 1. A method for producing a transgenic engineered cell that expresses a TEDV-encoded GOI from the TERS locus, comprising: a. Producing a TEDV encoding a GOI; b. delivering the TEDV to an engineered cell line containing a TERS paired with the TEDV, along with a recombinase enzyme that matches the RMCE element encoded by the TERS; c. contacting the cells with two or more affinity reagents specific for TEDV-encoded CST and TERS-encoded CST; d. Selecting the engineered cells based on the attenuation of expression of the reporter gene and CST encoded by TERS and the increase of expression of CST encoded by TEDV to select cells into which the GOI has been integrated. The present invention provides a method comprising:

[0009] In a third aspect, the present invention provides a tag exchange donor vector (TEDV) encoding a cell surface tag (CST) exon flanked by 3' intron fragments and a gene of interest (GOI) in the antisense orientation.

[0010] In a fourth aspect, the present invention provides an engineered cell comprising a tag exchange receiver site (TERS) in its genome, wherein the TERS encodes a cell surface tag (CST) exon and encodes a reporter gene in an antisense orientation, the CST exon being connected to an exon encoding a transmembrane domain by a full-length intron sequence, and wherein a recombinase-mediated cassette exchange (RMCE) element is contained in the TERS, and RMCE between the TERS and a tag exchange donor vector (TEDV) results in exchange of a gene of interest (GOI) encoded by the TEDV with the reporter element.

[0011] The present invention provides a system for integrating a GOI into an engineered cell line by RMCE, whereby the integration is conditionally detected by analysis of a co-integrated cell surface tag (CST). Furthermore, the concomitant loss of another CST from the engineered cell line upon integration of the GOI allows for double positive / negative selection of engineered cells expressing the GOI. This "tag swap" is highly robust and is the basis for the stringent positive / negative selection enabled by the CST system. A second selection gene is also lost from the TERS site, allowing for highly robust double-negative and single-positive selection during the generation of engineered cells expressing the GOI. Importantly, this nature of the CST system means that these markers are not only conditional, requiring the selective addition of a cognate affinity reagent for detection, but can also be exploited to physically partition target cell populations using a substrate-immobilized affinity reagent approach. This allows for an efficient, high-throughput method for generating engineered cells by partitioning cells using methods such as magnetic-activated cell sorting (MACS), as opposed to more time-consuming and difficult to parallelize methods such as fluorescence-activated cell sorting (FACS). Finally, instead of intracellular expression of fluorescent proteins and antibiotic resistance genes as markers of integration, the CSTE system offers a larger selection marker space. In particular, the creation of multiple unique CST epitopes for conditional selection is easily achieved, as opposed to fluorescent proteins with wide spatial overlap or a very limited set of antibiotic resistance genes. The broader access to selection markers enables different forms of high-throughput generation of engineered cells with increased efficiency, further enabling high-content methods for both cell library creation and cell lineage tracing in cell engineering, pathway engineering, and experimental workflows.

[0012] Detailed Description The overall structure and operation of the CSTE system is shown in Figure 1. The CSTE system operates as a donor / receiver pair, in which a tag exchange donor vector (TEDV) acts to deliver a GOI sequence and a cell surface tag (CST) exon to a tag exchange receiver site (TERS) paired with the TEDV, which is typically contained within the genome of an engineered cell line. The TERS encodes a different CST exon and a selected gene, which are exchanged with the TEDV-encoded CST and GOI, respectively. The TEDV-encoded CST exon is encoded in the sense (5' to 3') direction and in frame with a 3' intron fragment, which contains a splice acceptor site, a polypyrimidine tract, and a branchpoint sequence. A first RMCE element is encoded 5' to these functional intron sequences within a "non-functional" intron sequence. The GOI is encoded in the antisense (3' to 5') direction. A second RMCE element is encoded at the 3' end of the construct, adjacent to the GOI transcription start site.

[0013] The engineered cell line carries a TERS construct paired with TEDV that encodes a CST exon in frame with the full-length intron sequence and has a first RMCE element within the "non-functional" intron sequence, which is paired with and equivalent to the first RMCE element of TEDV. The full-length intron sequence encoding the first RMCE site contains a 5' splice donor site that is itself encoded in frame with the transmembrane domain (TD) exon. This TD contains a transcription initiation site and promoter sequence and drives 5' transcription of the TERS construct. Thus, the CST encoded by TERS is expressed on the cell surface upon translation from the spliced ​​transcript. Similarly, when RMCE occurs between TEDV and TERS, the CST exon encoded by TEDV is exchanged for the CST exon encoded by TERS. This results in "tag swapping," and the engineered cell line expresses TEDV-encoded CST on the cell surface, rather than TERS-encoded CST. In this process, the TEDV-encoded CST exon "gains" the 5' splice donor site, TD domain, transcription start site, and promoter sequence from the TERS construct, allowing expression of TEDV-encoded CST and providing strict site-specific control over integration of the TEDV-encoded sequence.

[0014] As described above, TERS encodes a selectable gene in the antisense (3' to 5') orientation, along with a promoter sequence capable of driving transcription of the selectable gene to the 3' end of the TERS construct. Between the transcription start site of the selectable gene in TERS and the 3' promoter sequence, a second RMCE element is encoded that pairs with the 25' RMCE element encoded in TEDV. Thus, when RMCE is performed between TEDV and TERS, the GOI encoded by TEDV is exchanged for the selectable gene encoded by TERS. It is important to note that because TEDV does not contain a promoter sequence, both the TEDV-encoded CST exons and the GOI must be supplemented with promoter sequences to enable their transcription. Importantly, as outlined above, including the intron and TD exon supplement for the CST exons provides even more rigorous site selectivity for integration of the TEDV-encoded sequence. The use of intron elements in the CSTE provides an additional level of assurance that random integration of the construct will not result in TEDV-encoded CST that can be expressed on the cell surface. In contrast to donor vectors that can deliver the full-length CST ORF, including the TD, the CSTE system delivers only the CST epitope exons and splice acceptor site. Thus, random, aberrant integration of the donor construct not only requires integration adjacent to an active promoter, but also requires acquisition of a type II transmembrane domain or its equivalent, along with an appropriate 5' splice donor sequence. This makes the occurrence of randomly integrated constructs expressing TEDV-encoded CST highly unlikely and minimizes the selection of such random, unwanted events during the generation of derivative engineered cells expressing the delivered GOI. The specific structures of the CSTE system components, including TEDV and TERS, are detailed below.

[0015] TERS structure The engineered cell line contains TERS, which constitutively expresses the TERS-encoded CST and a selected gene, which are exchanged for the TEDV-encoded CST and GOI, respectively, upon RMCE with the TERS-paired TEDV. The gene structure of TERS is shown in Figure 3. A TERS typically includes: a) Kozak sequence b) Exons encoding the transmembrane domains of type 2 membrane proteins c) 5' intron splice donor site d) a 5' RMCE element encoded within a "non-functional" non-coding 3' intron fragment that is an equivalent of and pairs with the 5' RMCE element of TEDV; e) Functional sequence of the 3' intron fragment, including the branch point sequence, polypyrimidine tract, and splice acceptor site f) An exon containing CST encoded by TERS in the 5' to 3' direction g) a transcription terminator sequence for CST encoded in the 5' to 3' direction h) a transcription terminator sequence for the selected gene encoded 3' to 5' i) a sequence encoding a selection gene in the 3' to 5' direction j) Kozak sequence for efficient translation initiation of transcripts of selected genes k) 3' RMCE element x) A 5' genomic element responsible for regulating expression and tracking of the CST transcript. This genomic element minimally contains the promoter sequence that drives CST transcription. y) A 3' genomic element responsible for regulating expression and tracking of the selected gene transcript. This genomic element minimally contains a promoter sequence that drives transcription of the selected gene. Here, a) is provided to ensure efficient translation initiation from the tethered TD / CST transcript driven from TERS for both the CST encoded by TERS before RMCE and the CST encoded by TEDV after RMCE. b) is a TD that is tethered to a CST exon by transcript splicing, mediates insertion of the translated protein construct into the cell membrane, and presents CST in the extracellular space. d) is an RMCE element located within the intron sequence of the TERS construct, equivalent to the 5' RMCE element of TEDV, and mediates CST exon exchange during RMCE in the CST system. e) is an intronic splice acceptor site and associated functional sequence that enable splicing of the intron containing the RMCE element from the TD / CST transcript driven from TERS. f) is an exon within TERS that encodes a CST epitope, and the expressed TD / CST protein presents the epitope in the extracellular space, which can be conditionally detected using a cognate affinity reagent. g) is a transcription terminator for the TD / CST open reading frame encoded in the 5' to 3' direction. h) is a transcription terminator for the selection gene open reading frame encoded in the TERS in the 3' to 5' direction. i) is an open reading frame encoding the selection gene to be expressed from TERS in the engineered cell line. j) is a Kozak sequence provided to ensure efficient translation initiation from the selection gene transcript driven from TERS before RMCE. k) is an RMCE element, equivalent to the 3' RMCE element of TEDV, located between the transcription start site of the selection gene and the promoter sequence driving transcription of its open reading frame. It mediates the exchange of the TEDV-encoded GOI and the selection gene during RMCE in the CSTE system. x) is a promoter sequence that, at a minimum, drives transcription of the TERS-encoded TD / CST open reading frame before RMCE and the TEDV-encoded TD / CST after RMCE.y) is, at a minimum, a selected gene open reading frame encoded by TERS before RMCE and a promoter sequence driving transcription of a GOI encoded by TEDV after RMCE. A schematic diagram of the TERS cloning fragment is shown in FIG.

[0016] TERS essentially encodes a fully functional intron sequence containing a 5' RMCE site. Such a sequence can be any intron that allows efficient splicing of the TD / CST transcript in the host engineered cell. The splice acceptor site of TEDV must be functional in cooperation with the splice donor site of TERS. Therefore, the splice acceptor sequences encoded by TEDV and TERS should be identical or equivalent. The "non-functional" non-coding intron sequence encoding the RMCE site may be used to encode additional genetic elements, such as transcriptional enhancers, transcriptional insulators, other open reading frames encoding other desired components for function or reporting of the TERS construct in engineered cells, or other unique sequences used to trace and quantitate transcripts and / or constructs. The sequence may be encoded. With respect to TERS, both the encoded TD / CST and the selection gene may contain 5' and 3' untranslated regions (UTRs) that encode unique sequences used for tracing and quantitation of transcripts and / or constructs or that confer, for example, regulation of transcript stability.

[0017] The selection gene can be chosen from the following: a. antibiotic resistance genes, b. a reporter gene; c. auxotrophic complementing genes, D. Inducible suicide gene. The selection, format, and application of such positive selection markers are well known to those skilled in the art. The use of an inducible suicide gene can be used to eliminate the parent engineered cells after RMCE in the generation of derivative engineered cells expressing the GOI. The reporter gene can be a unique TD / CST construct for conditional or constitutive reporting at the cell surface. Multiple selection genes can be included in this portion of the TERS to allow for positive selection during the generation of engineered cell lines and negative selection during the generation of derivative engineered cell lines.

[0018] The 5' and 3' genomic elements encode, at a minimum, promoter sequences that mediate transcription of the TD / CST and selected genes. Such promoters can be constitutive or inducible. These genomic elements may also encode additional genetic elements, such as transcriptional enhancers, transcriptional insulators, additional open reading frames encoding other desired components for function or reporting of the TERS construct in engineered cells, or other unique sequences used to trace and quantitate transcripts and / or constructs. It is important to note that, herein, the TD / CST is encoded in the sense (5' to 3') orientation and the selected gene is encoded in the antisense (3' to 5') orientation (as shown in Figure 3). This orientation is for clarity of illustration. With respect to TERS / TEDV, there is no practical reason why the orientation could not be reversed, provided that the TD / CST construct also incorporates a functional intron sequence that encodes an equivalent RMCE site that pairs with the RMCE site in the 3' intron fragment of TEDV. The inclusion of a selection gene is not essential for the operation of the CSTE system, but is desirable because it serves as a triple selection for GOI expression after RMCE. Furthermore, by generating and maintaining engineered cell lines with reporters in both sense and antisense orientations, we can ensure that the promoter sequences contained within the 5' and 3' genomic elements are sufficiently functional to generate derivative engineered cells that express the TEDV-encoded GOI prior to RMCE.

[0019] Cell surface tag (CST) structure One of the key functional features of the CSTE system is the use of multiple unique CST constructs that conditionally report the presence of the original TERS construct and the exchanged construct that incorporates TEDV-encoded sequences via RMCE. To achieve TD recruitment in the CSTE system, only certain types of TD exons are suitable: those that can be spliced ​​together with the 3'-located CST exon while also exposing a CST protein fragment extracellularly. To simplify the expressed CST epitope-bearing protein product and minimize the size and complexity of the TD exon, a type II membrane protein TD is most suitable. Type II TDs span the membrane once and are located at the N-terminus of the protein on the cytoplasmic side of the membrane, allowing the CST epitope at the C-terminus to be exposed extracellularly. Other TDs, including multi-spanning TDs, may also be used, provided that the spliced ​​CST transcript exposes the CST epitope to the extracellular space.

[0020] The CST exons that are joined to the TD in the spliced ​​transcript simply function to present unique epitopes extracellularly that can be conditionally detected by the use of cognate affinity reagents. Typically, such epitopes include synthetic sequences, sequences encoded in an organism other than the host engineered cell's organism, or sequences from the same organism as the host engineered cell but that are not expressed extracellularly. Generally, it is desirable to include a linker domain between the TD and the CST epitope. Because the CST epitope can consist of only a few amino acids, such a linker region ensures that CST is available on the cell surface for affinity reagent binding. Such a linker region may comprise a flexible, "unstructured" region or a structured, fully folded protein domain. The nature of the epitopes encoded by the CST exons reflects only their uniqueness in the context of epitopes otherwise expressed on the cell surface of engineered cells containing TERS, and thus may represent any protein sequence for which a specific affinity reagent can be raised. In general, the CST construct as a whole should be functionally neutral with respect to cellular function. Often, a transmembrane domain simply fused, via a linker sequence, to an inert epitope structure without any exogenous functional domains is preferred.

[0021] In the context of the present invention, an affinity reagent is defined as any antibody, peptide, nucleic acid, or other small molecule that specifically binds to a larger target molecule to identify, track, capture, or affect the activity of a CST epitope. Frequently, such affinity reagents are labeled with fluorescent, colorimetric, radiometric, or other detectable labels to track cells expressing the cognate CST. Alternatively, such affinity reagents may be functionalized onto substrates, enabling substrate affinity enrichment methods for distributing engineered cells expressing CST.

[0022] Engineered cell lines containing TERS A cell line containing a TERS is engineered because it has a synthetic TERS construct inserted into it. Generally, this refers to integration of the TERS construct into the genome of the engineered cell, but may also include other methods for nuclear maintenance of the TERS construct, such as methods for episomal maintenance of gene constructs. Methods for integrating constructs into the genome of target mammalian cells are well known to those of skill in the art and may be achieved by homologous recombination (HDR) and / or random integration methods, where HDR may be facilitated by targeted mutation of the genomic locus at which HDR occurs, including but not limited to: i. Zinc finger nuclease ii.CRISPR / Cas9-mediated targets iii. Synthetic Transcription Activator-Like Effector Nucleases (TALENs) This can be achieved by a variety of means, including site-specific mutagenesis by HDR, where the site-specific nuclease induces site-specific DNA repair by HDR at the target locus. After this event, a proportion of cells will have integrated the HDR vector and can be selected and / or determined by any combination of the following: iv. Non-destructive phenotypic expression analysis v. Disruptive phenotypic expression analysis vi. Genetic analysis Here, iv and vi are the preferred methods for selecting and determining the success of genomic integration events. Alternatively, viral vectors can be used to deliver the necessary components in a site-specific or non-specific manner.

[0023] The methodology of TERS construct integration is not central to TERS operation, so long as there is detectable TERS-encoded CST at the cell surface and the selection gene is expressed (indicating that the construct is functional with respect to transcription of the included coding sequence). Indeed, the TERS-encoded CST and the selection gene together are convenient selection markers for the generation of engineered cell lines containing TERS. One important aspect to note is that control of the copy number of the integrated TERS construct is required for some applications, particularly those that exploit the ability to generate cell-based arrays of engineered cell lines expressing a single GOI from a library of TEDV constructs, and similar "multiplexing" methods. In this regard, episomal maintenance of TERS constructs is largely incompatible with such multiplexing methods for generating engineered cell lines. Methods for determining construct copy number within the genome of engineered cells are well known to those skilled in the art.

[0024] Structure of TEDV TEDV encodes the CST exons and GOI that are exchanged with the TERS-encoded CST exons and selection gene, respectively, contained in the engineered cell line during RMCE. The general structure of TEDV is shown in Figure 2. TEDV typically includes: 1) A 5' RMCE element encoded within a "non-functional" non-coding 3' intron fragment 2) A 3' intron fragment containing a branch point sequence, a polypyrimidine tract, and a 3' splice acceptor site. 3) An exon containing the CST encoded by TEDV in the 5' to 3' direction 4) A transcription terminator sequence for CST encoded in the 5' to 3' direction 5) 3'→5' encoded transcription terminator sequence for the GOI 6) Sequence encoding GOI in the 3'→5' direction 7) Kozak arrangement 8) 3' RMCE element. Here, 1) is a RMCE element located within the nonfunctional, noncoding intron sequence of the 3' intron fragment of the TEDV construct and is equivalent to the 5' RMCE element of TERS, which mediates CST exon exchange during RMCE in the CSTE system. 2) is a 3' intron fragment that provides a branchpoint sequence, polypyrimidine tract, and 3' splice acceptor site for efficient splicing of the integrated TEDV-encoded CST exon into the TD / CST transcript after RMCE between TEDV and TERS to generate a derivative-engineered cell expressing the GOI. 3) is an exon within the TEDV construct that encodes a CST epitope. The complemented and expressed TD / CST protein after RMCE between TEDV and TERS presents the epitope in the extracellular space, where it can be conditionally detected using a cognate affinity reagent. 4) is a transcription terminator for the CST exon encoded in the 5' to 3' direction, which acts as a transcription terminator for the TD / CST complemented by the CST encoded by the TEDV upon RMCE. 5) is a transcription terminator for the GOI open reading frame encoded in the 5' to 3' direction within the TEDV, which acts as a transcription terminator for the GOI coding sequence integrated into the TERS site upon RMCE. 6) is an open reading frame encoding the GOI to be integrated into the engineered cells to generate a derivative engineered cell that expresses the GOI. 7) is provided to ensure efficient transcription initiation of the GOI transcript resulting from the TERS after RMCE. 8) is an RMCE element at the 3' end of the TEDV construct, equivalent to the 3' RMCE site of the TERS, located between the transcription start site of the selected gene and the promoter sequence driving transcription of its open reading frame. A schematic diagram of the TEDV cloning fragment is shown in FIG.

[0025] The TEDV may be a synthetic DNA construct or a cloned construct generated by methods well known to those of skill in the art. To aid in cloning procedures, the TEDV may contain restriction endonuclease sequences for inserting various CST exons and / or GOI sequences into the construct. The inclusion and use of such cloning sites is well known to those of skill in the art.

[0026] In the context of the present invention, TEDV is generally a plasmid construct that is propagated in bacteria and may also contain an origin of replication and a selection gene. The TEDV may be a construct that contains sequence motifs that allow the construct to be packaged into a specific delivery vector, such as a viral vector known to those skilled in the art. The use of viral vector transduction of engineered cells containing TEDVs would be beneficial for the use of the CSTE system in difficult-to-transfect cells. The TEDV may be an RNA construct that can be reverse transcribed by providing an appropriate reverse transcriptase.

[0027] In the context of the present invention, a GOI is defined as any coding or non-coding sequence of interest, which may include any protein- or polypeptide-open reading frame, non-protein-coding RNA, such as microRNA, small hairpin RNA, tRNA or rRNA. Importantly, a GOI can be a library of variants of a single open reading frame, and TEDVs encoding such GOIs can be followed by pooled libraries of TEDVs. Because the CSTE system allows precise control of copy number and reliable, multifactorial, and conditional reporting of GOI integration, it is possible to generate high-content libraries of engineered cells that express only a single GOI in each engineered cell in a pool of target cells. This can be exploited to engineer a variety of cells, pathways, proteins, and specific GOIs in the context of live cells (see below).

[0028] RMCE Elements and Enzymes The use of site-specific recombinases (SSRs) has proven to be a promising tool for modifying cellular genomes. There are two major classes of SSRs: Ser integrases, including but not limited to ΦC31, γδ-res, ParA, Tn3, Gin, ΦBT1, R4, Bxb1, and TP901-1; and Tyr recombinases, including but not limited to Flp, Cre, and R. While both classes can perform RMCE, Ser integrases are limited by their reliance on the initial crossover, resulting in the introduction of either the desired TEDV-encoded CST and GOI or the TEDV vector backbone. While not all Tyr recombinases have been comprehensively evaluated, Cre and Flp have been widely used to perform RMCE. For the purpose of integrating a single copy of the TEDV-encoded CST and GOI genetic elements, the use of Flp with a well-characterized heterologous FRT site is optimal because there are no pseudo-FRT sites encoded in the human genome. This is not the case for Cre, which exhibits promiscuous activity on LoxP genomic pseudosites.

[0029] Methods for producing engineered cells The use of RMCE in mammalian cells is well known to those skilled in the art, where a donor construct can be delivered to a target cell by a variety of methods, including, but not limited to, chemical transfection, electroporation, or viral vector delivery. In addition to providing the donor construct, a specific recombinase enzyme must also be provided. This is usually provided as a separate expression construct that is co-delivered to the target cell along with the donor construct. In some cases, it may be desirable to modify engineered cells expressing TERS to conditionally express the required recombinase enzyme, increasing the efficiency of the overall process. In the context of the CSTE system, once RMCE is performed by delivery of TEDV to engineered cells containing TERS, following a growth period, the target cell population can be analyzed or selected for generation of derived engineered cells expressing the TEDV-encoded GOI. This can be accomplished by three methods that are unique to the CSTE system and do not rely on detection of GOI expression. Derived engineered cell lines can be selected based on negative selection of TERS-encoded CST and / or positive selection of TEDV-encoded CST, reflecting successful tag exchange. These conditionally report and require the addition of cognate affinity reagents for each CST. Such selection methods can utilize FACS and selection of the desired CST expression profile by addition of fluorescently labeled affinity reagents. Because CST epitopes are extracellularly displayed, cell partitioning can also be achieved by substrate-based enrichment approaches such as MACS.

[0030] Further negative selection can be achieved based on the TERS-encoded selection gene, where the selection gene is a reporter gene or an inducible suicide gene. The reporter gene can utilize FACS, for example, in the case of a fluorescent reporter gene, or FACS and / or MACS if the reporter gene itself is a unique CST. The inducible suicide gene can be used to negatively select parent engineered cells from culture to enrich for derivative engineered cells expressing the GOI. Overall, the double-positive and single-negative selections provide a highly robust selection of engineered cells that can be conditionally reported and used for cell partitioning using substrate-immobilized affinity reagents. This selection may further include positive selection for a TEDV-encoded GOI if the nature of the GOI is amenable to nondestructive detection in the engineered cells. The above selections can be used serially or in parallel to achieve high-throughput or accurate, high-content engineered cell populations.

[0031] These above-mentioned phenotypic analyses are usually supported by genotypic confirmation of the copy number of TEDV sequences in the derived engineered cells using methods well known to those skilled in the art. This may be achieved phenotypically to the extent that, given the highly standardized nature of CST expression, the extremely rare cases in which aberrant integration of a TEDV construct results in expression of TEDV-encoded CST at the cell surface can be negatively selected for by elimination of cells with high TEDV-encoded CST expression, particularly when FACS selection methods are used. In the case of engineered cells expressing multiple TERS sites (see below), copy number control can similarly be achieved based on the degree of TEDV-encoded CST expressed at the cell surface.

[0032] Cell partitioning using substrate-immobilized affinity reagents A key advantage of the CSTE system is the cell surface exposure of the CST epitope, thereby enabling cell partitioning based on a substrate-immobilized affinity method. This is particularly useful for high-throughput methods or for stepwise selection in the generation of high-content derivative engineered cell libraries. A method well known to those skilled in the art is the MACS approach, based on ferromagnetic beads, among other substrates functionalized with various other affinity reagents, for the capture and physical partitioning of cells based on specific expression of surface epitopes. Such rapid, conditional positive and / or negative selection based on cell surface CST expression is highly parallelizable and therefore suitable for high-throughput methods, is much faster and more accurate than antibiotic resistance selection, and is much faster and less costly than FACS selection, which is limited to intracellular fluorescent proteins. Furthermore, the ability to positively and negatively select both parental and derivative engineered cells leads to the generation of accurate, high-content libraries of derivative engineered cells expressing one or more GOIs.

[0033] Cell surface "barcoding" methods using multiple and multivalent CSTs An advantage of the CSTE system is the increased amount of available selectable marker space. In contrast to standard fluorescent protein reporter and antibiotic resistance gene systems, which offer only a handful of parallelizable markers, many unique markers can be rapidly generated by using simple epitopes with cognate affinity reagents. One is not limited solely by the number of available CST epitopes and cognate reagents, because multi-epitope CST domains can be readily constructed for every unique combination of available CST epitopes and cognate detection reagents. The expanded, parallelizable conditional reporter space enables a variety of high-throughput, high-content methods. The use of multiple TEDV-encoded CST epitopes, each associated with a different GOI or GOI variant sequence, can increase the efficiency of high-throughput cell engineering. For example, a pool of such unique vectors can be combined into a pool of engineered cells expressing TERS. These can be handled together during negative selection, as outlined above, and later distributed based on unique TEDV-encoded CST expression. Similarly, the use of multiple CST epitopes encoded by TEDV allows for the generation of medium-content libraries for tracking and selection of viable cell lineages. For example, libraries of unique CST-associated GOIs can be incorporated into pools of engineered cells, and the persistence or function of these cell lineages expressing specific GOIs can be traced over time. This has applications in selection in culture systems for protein, pathway, or cell engineering or analysis, or may be used similarly when cells are passaged into live animals and later recovered as viable cells for analysis.

[0034] High-content libraries and multi-TERS engineered cells Robust positive and negative selection of engineered cells expressing a GOI allows for the generation of accurate high-content libraries in multiplexed formats to support protein, pathway, and cell engineering workflows. In the simplest concept, a TEDV library encoding a single CST epitope but multiple GOIs can be integrated into a pool of cells. Application of standard positive / negative CST selection using TERS and TEDV-encoded CST, for example, will lead to a population of engineered cells expressing a single GOI variant in each cell of the selected population. These high-content cell libraries can then be subjected to functional selection in a workflow to engineer proteins, cellular pathways, or more generally, cellular functions based on the integrated variant GOIs. In addition to multiple TERS containing unique CST epitopes in a single engineered cell and multiple TEDV libraries containing unique CST epitopes, multiple families of variant GOIs may be incorporated into such an engineered workflow, e.g., treating enzymes in a biosynthetic pathway as combinatorial variant GOIs. This situation, in which engineered cells contain multiple TERS, can be used to facilitate global expression of a single GOI. Multi-TERS engineered cells typically require the use of a unique heterospecific recombinase for each TERS / TEDV pair to ensure efficiency and stability of GOI integration and selection. [Brief explanation of the drawings]

[0035] [Figure 1]Composition and Operation of the Cell Surface Tag Exchange (CSTE) System. Schematic diagram of the CSTE system. The top panel shows system components, including a tag exchange donor vector (TEDV) (left) and engineered cells containing a tag exchange receiver site (TERS) (right). TEDV encodes RMCE elements (open triangle and filled triangle) at both ends of the construct, pairing with the RMCE site contained in TERS. The RMCE element (filled triangle) at the 5' end of the TEDV construct is encoded within a sequence that is a 3' intron fragment, and immediately 3' to the RMCE element is the 3' intron element (open circle), which includes a branchpoint sequence, a polypyrimidine tract, and a splice acceptor site. Thus, RMCE is contained within a "non-functional," non-coding intron sequence, and the 3' intron fragment contained in TEDV lacks a 5' splice donor site. Immediately 3' from the splice acceptor site, TEDV encodes the 3' exon of the cell surface tag (CST). This means that the exon encodes the portion of CST containing a unique molecular binding motif (gray rectangle). The CST sequence is encoded in the 5' to 3' direction. TEDV also encodes a gene of interest (GOI) (hatched rectangle) that is integrated into TERS, encoded in the 3' to 5' direction. The central portion of TERS contained in engineered cells encodes a similar structure but distinct elements from those in TEDV. That is, TERS encodes a CST exon (checkered rectangle) distinct from the TEDV-encoded CST between the RMCE element (open triangle and filled triangle) that is paired with that of TEDV, and immediately 5' from this CST contains the splice acceptor site and associated 3' intron sequence (open circle). Similarly, a selection gene (filled rectangle) is encoded in the antisense direction within TERS, similar to the GOI in TEDV. The 5' end of the construct contains a promoter sequence (arrow pointing right) that drives transcription of CST, and 3' to this promoter sequence is an exon (open rectangle) encoding the transmembrane domain (TD), with a 5' intron sequence (filled circle) immediately 3' from that.This means that the TD and CST exons, encoded in frame with the RMCE element-containing intron, are generated as a continuous transcript that is spliced ​​to join the exons into a single coding mRNA. The TERS-encoded TD-CST product (open / checkered dumbbell shape) is expressed on the cell surface. At the 3' end of the TERS construct is another promoter element that drives transcription of the selection gene (filled rectangle) in the 3' to 5' direction, resulting in the expression of the selection gene (filled square). Introducing TEDV into engineered cells containing TERS, along with an appropriate expression construct for a recombinase specific for the paired RMCE element in TEDV / TERS, results in RMCE and generates derivative engineered cells expressing the GOI (bottom panel). The TERS-encoded element is replaced by a TEDV-encoded element. Thus, the derived cell line expresses TEDV-encoded CST on the cell surface as a TD-CST product with the original TERS-encoded TD (open / gray dumbbell shape) and expresses the GOI (hatched square). In summary, RMCE between TEDV and TERS results in the loss of expression of the selected gene and TERS-encoded CST, generating a derived cell line that acquires expression of TEDV-encoded CST and the GOI. [Figure 2]Structure of the Tag Exchange Delivery Vector (TEDV). Schematic diagram of the TEDV shown as a linear construct, with each numbered box representing a key element of the construct's structure. The construct contains both the TEDV-encoded CST and gene of interest (GOI). 1) represents the 5' RMCE element encoded within the "nonfunctional" noncoding 3' intron fragment. 2) represents the functional sequence of the 3' intron fragment, including the branchpoint sequence, polypyrimidine tract, and 3' splice acceptor site. 3) represents the exon encoding the TEDV-encoded CST in a 5' to 3' direction. 4) represents the transcription terminator sequence for the CST encoded in a 5' to 3' direction. 5) represents the transcription terminator sequence for the GOI encoded in a 3' to 5' direction. 6) represents the sequence encoding the GOI in a 3' to 5' direction. 7) represents a Kozak sequence for efficient translation initiation of the GOI transcript. 8) represents the 3' RMCE element. [Figure 3]Tag exchange receiver site (TERS) open structure - before tag exchange. Schematic diagram of TERS shown as a linear construct, with each lettered box representing a key element of the construct's structure. The construct contains both the TERS-encoded CST and a selection gene. a) Represents a Kozak sequence for efficient translation initiation of the tethered transmembrane domain (TD) / CST transcript. b) Represents an exon encoding a type 2 membrane scaffold protein domain. c) Represents a 5' intron splice donor site. d) Represents a 5' RMCE element encoded within a "nonfunctional" noncoding 3' intron fragment that is equivalent to and pairs with the 5' RMCE element of TEDV. e) Represents the functional sequence of the 3' intron fragment, including the branchpoint sequence, polypyrimidine tract, and 3' splice acceptor site. f) Represents an exon encoding the TERS-encoded CST in the 5' to 3' direction. g) represents a transcription terminator sequence for CST encoded in the 5' to 3' direction. h) represents a transcription terminator sequence for a selected gene encoded in the 3' to 5' direction. i) represents a sequence encoding a selected gene in the 3' to 5' direction. j) represents a Kozak sequence for efficient translation initiation of the selected gene transcript. k) represents a 3' RMCE element. x) represents a 5' genomic element responsible for regulating and tracking the expression of the CST transcript. This genomic element minimally comprises a promoter sequence that drives CST expression. y) represents a 5' genomic element responsible for regulating and tracking the expression of the CST transcript. This genomic element minimally comprises a promoter sequence that drives CST expression. [Figure 4] TERS after Exchange in Derived Engineered Cells. Schematic diagram of the TERS locus after RMCE-mediated exchange with TEDV-encoded elements. As shown in Figure 1, the elements encoded between the RMCE sites of TEDV are exchanged with sequences of equivalent structure encoded between the RMCE sites of TERS. This results in exchanged TERS in the engineered cell line. Lettered and numbered boxes represent key genetic elements detailed in Figures 2 and 3, respectively. [Figure 5]Schematic representation of the TEDV by-product after RMCE-mediated exchange with a TERS-encoded element. As shown in Figure 1, the element encoded between the RMCE sites of TEDV is exchanged with an equivalent structural sequence encoded between the RMCE sites of TERS. This results in a post-exchange TEDV by-product that is unstable during the generation of the derivative engineered cell line. Lettered and numbered boxes represent key genetic elements detailed in Figures 2 and 3, respectively. [Figure 6] Integration of TERS into engineered cells. a) The engineered cell line population, ACL-1163, was created by homologous recombination of a TERS cassette encoding the Myc epitope CST and RFP selection genes into the parental cell line, ACL-128. Ten days after electroporation, cells were stained with anti-Myc antibody and analyzed by flow cytometry for the TERS-encoded selectable markers, Myc epitope CST and RFP. The plot shows surviving single cells as RFP vs. Myc, demonstrating the presence of a transfectant population (Q2 left panel) that exhibits higher RFP and Myc signals compared to the parental cells (Q2 right panel). b) Cells with high RFP and Myc signals were selected and expanded, and a representative engineered monoclone, ACL-1163, was analyzed by flow cytometry. The plot shows the RFP vs. Myc parameters for the gated surviving single cells. Monoclone ACL-1163, as expected, has high RFP and Myc signals (Q2). [Figure 7]Generation of Derived-Engineered Cells by RMCE with TEDV with Tag Exchange and GOI Delivery. Flp recombinase-mediated tag exchange was performed in ACL-1163 engineered cells. ACL-1163 cells carrying a TERS encoding the RFP selection gene and the Myc epitope CST were co-transfected with TEDV encoding the SBP epitope CST and GOI, along with a construct encoding flp recombinase. Seven days after electroporation, cells were stained with anti-Myc and anti-SBP antibodies and analyzed by flow cytometry for RFP, Myc, and SBP signals. Cells with reduced RFP and Myc signals but high SBP surface expression signals were selected and expanded as monoclones. a and b) Contour plots showing a representative derived-engineered cell monoclone, ACL-3426 (left), compared with parental cells (right). Derived cells show loss of RFP and Myc signals (Q4 upper left panel) compared with parental cells (Q4 upper right panel). The derived engineered cell monoclone ACL-3426 successfully expressed the TEDV-encoded SBP epitope CST, as indicated by an increased signal when stained with an anti-SBP antibody (Q5 lower right panel). These results suggest that the Flp recombinase-mediated tag exchange was successful, as ACL-3426 cells lost RFP and Myc marker signals and acquired SBP surface expression, as expected for the CST tag exchange between TERS and TEDV. c) Immunoblot showing three examples of expression of GOI integrated with a C-terminal FLAG tag. Detection of GOI (RSV-1 ORF) expression was achieved by immunoblotting using an antibody against the Flag tag. The parental monoclonal line ACL-1163 was included as a control.CST was performed in three independent experiments. Cells were transfected with constructs encoding flp recombinase and either TEDV encoding the SBP epitope CST and the RSV-1 P gene (resulting cell line monoclone: ​​ACL-3374), TEDV encoding the SBP epitope CST and the RSV-1 N gene (resulting cell line monoclone: ​​ACL-3386), or TEDV encoding the SBP epitope CST and the RSV-1 M2 long gene (resulting cell line monoclone: ​​ACL-3433). Proteins were extracted from the monoclonals and immunoblotted using a mouse anti-Flag antibody. Western blot results demonstrated that each RSV-1 ORF was expressed, as evidenced by the presence of a single band corresponding to the expected molecular weight of each GOI in the corresponding cell line and the absence of a signal in the parental cell line. [Figure 8]Enrichment of Tag2 (SBP) or depletion of Tag1 (Myc) from a mixed cell population after tag exchange by magnetic-activated cell sorting (MACS). This figure demonstrates that surface tag technology can be used for MACS enrichment of Tag2-bearing cells or depletion of Tag1-bearing cells from a mixed cell population after tag exchange. Two engineered monoclonal cell lines, APL-4535 and APL-3015, were used as the starting mixed population to enrich for Tag2 (SBP)-expressing cells or deplete Tag1 (Myc)-expressing cells after the tag exchange event by MACS. The TERS in APL-4535 cells encoded the SBP epitope CST and a full-length FLAG-tagged GOI encoding an intracellular protein, while the TERS in APL-3015 cells encoded the Myc epitope CST and an RFP selection gene. The two cell populations were mixed at a ratio of 90% (APL-3015 cells) to 10% (APL-4535 cells) and then MACS was used to enrich for SBP-positive cells (a) or deplete Myc-positive cells in separate experiments (b). a) All cells were labeled with anti-SBP-Alexa 488 fluorophore and incubated with anti-mouse IgG iron beads. Samples were run through MACS, and fractions were collected at three experimental time points: pre-enrichment, flow-through, and bound cells. All three fractions were counterstained with anti-c-Myc-Alexa 405, and data were acquired using a BD Influx instrument. Graphs show the percentages of SBP-positive and Myc-positive cells in all three experimental fractions. The pre-MACS fraction shows that the starting cell population consisted of 90% Myc-positive and 10% SBP-positive cells. Successful enrichment of SBP-positive cells was indicated by the absence of SBP signal in the flow-through fraction (>0.01% SBP-positive cells), whereas 95% of bound cells were SBP-positive after SBP-targeted enrichment. b) In a separate experiment, all cells were labeled with anti-c-Myc-Alexa 405 fluorophore and incubated with anti-mouse IgG iron beads. Labeled cells were depleted using MACS, and fractions were collected at the three experimental time points indicated above. All three fractions were counterstained with anti-SBP-Alexa 488, and data were acquired on a BD Influx instrument. Graphs show the percentages of SBP-positive and Myc-positive cells in all three experimental fractions.MACS prefractionation again showed that the starting cell population consisted of 90% Myc-positive cells and 10% SBP-positive cells. Successful depletion of Myc-positive cells was indicated by a reduction in the number of Myc-positive cells (25%) and an increase in the number of SBP-positive cells (75%) in the flow-through fraction. Furthermore, the combined fraction contained >90% Myc-positive cells and <5% SBP-positive cells. c) A total of 546 SBP-positive staining monoclones were individually evaluated for encoding a GOI linked to SBP. The figure shows that 96.52% of SBP-positive cells had integrated the GOI, while 3.48% of SBP-positive cells did not. These results demonstrate that MACS can be used to enrich for Tag2(SBP)-positive cells or deplete Tag1(Myc)-positive cells from mixed cell populations. Furthermore, the presence of Tag2(SBP) can be used as an indicator of successful tag exchange and GOI genomic integration. [Figure 9]Construction and operation of the barcode-based cell surface tag exchange (CSTE) system. Schematic diagram of the barcode-based CSTE system. (a) shows the components of the tag exchange donor vector (TEDV). Each TEDV encodes an RMCE element (open triangle and filled triangle) at both ends of the construct, pairing with the RMCE site contained in the TERS. The RMCE element (filled triangle) at the 5' end of the TEDV construct is encoded within the 3' intron fragment sequence. Immediately 3' of this RMCE element is an intronic 3' element (open circle) containing the branch point sequence, polypyrimidine tract, and splice acceptor site. Immediately 3' of the splice acceptor site, the TEDV encodes the 3' exon of the cell surface tag (CST). This means that this exon encodes the portion of CST (gray rectangle) containing the unique molecular binding motif. The CST sequence is encoded in the 5' to 3' direction, while the TEDV also encodes a gene of interest (GOI) (hatched rectangle) that is integrated into the TERS, encoded in the 3' to 5' direction. Each CST in this example is composed of two epitopes (A, B, and C) selected from three unique epitopes (six unique combinations possible). AB is functionally equivalent to BA. In this example, the AX combination was assigned to a GOI family with three variants (GOI ai, GOI a-ii, and GOI a-iii), and the BX combination was assigned to a second GOI family with three variants (GOI bi, GOI b-ii, and GOI b-iii). The individual TEDVs in this example are pooled. (b) shows the components of the tag exchange receiver site (TERS). The TERS RMCE element pairs with the RMCE element of the TEDV (open and filled triangles). TERS encodes a distinct CST exon (checkered rectangle) from the TEDV-encoded CST exons, and a splice acceptor site and associated 3' intron sequence (open circle) are located immediately 5' to this CST exon. Similarly, a selection gene (solid rectangle) is encoded in the antisense orientation within TERS, similar to the GOI in TEDV. The 5' end of the construct contains a promoter sequence (right-pointing arrow) that drives transcription of CST.3' to this promoter sequence encodes the transmembrane domain (TD) exon (open rectangle), which is immediately 3' to the 5' intron sequence (filled circle). The TERS-encoded TD-CST product (open / checkered dumbbell shape) is expressed at the cell surface. At the 3' end of the TERS construct is another promoter element that drives transcription of a selection gene (filled rectangle) in the 3' to 5' direction, resulting in an expressed selection gene (filled square). Figure c) shows that introduction of a TEDV pool into an engineered cell population containing TERS, along with an appropriate expression construct for a recombinase specific for the paired RMCE element in TEDV / TERS, results in RMCE, generating derived engineered cell populations expressing various GOIs. During RMCE, the TERS-encoded element is exchanged for the TEDV-encoded element. Thus, the derived cell population expresses TEDV-encoded CST on the cell surface as a TD-CST product, with the original TERS-encoded TD (open / gray dumbbell shape), and expresses the GOI (hatched square). d) After RMCE, the pool of engineered cells expressing the GOI can be further isolated into individual members by FACS based on the expression of unique CST barcodes. This can be achieved as a bulk population of the desired barcode or by individual cell isolation using single-cell sorting methods. Alternatively, analysis of the pool can be performed by FACS without sorting, using the barcode as a means to identify the population of interest within the digital dataset. e) To prove the principle of barcoding engineered cells using the CSTE system, ACL-5 and ACL-1 cells were transfected with either a CST-encoding plasmid or a CST-free control plasmid by chemical transfection (ACL-5) or electroporation (ACL-1) using standard methods known to those skilled in the art. This CST contained three unique epitopes: FLAG, MYC, and HA (as shown in SEQ ID NO: 20). 48 hours after transfection, cells were harvested and stained with fluorophore-conjugated cognate antibodies: anti-FLAG-PE, anti-MYC-AF647, and anti-HA-AF488. Cells were analyzed by flow cytometry.Viable cells were gated by forward scatter (FSC) and side scatter (SSC). The mean fluorescence intensity (MFI) of viable cells was determined for each of the three epitopes, and the percentage of viable cells expressing each epitope was determined. All three epitopes were detected at a higher rate and intensity in cells transfected with a plasmid encoding barcoded CST compared to cells transfected with an empty vector. This demonstrates the ability of CST to be composed of multiple epitopes. DETAILED DESCRIPTION OF THE INVENTION

[0036] The following list of non-limiting embodiments further describes the present invention. 1. A combination system comprising two separate components: a first component is a tag exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon flanked by 3' intron fragments and a gene of interest (GOI) in an antisense orientation; a second component is an engineered cell containing in its genome a tag exchange receiver site (TERS), the TERS encoding a second CST exon and encoding a reporter gene in an antisense orientation, the second CST exon being connected by a full-length intron sequence to an exon encoding a transmembrane domain; Here, paired recombinase-mediated cassette exchange (RMCE) elements are contained in TEDV and TERS, and RMCE between TEDV and TERS results in exchange of the GOI encoded by TEDV with a reporter element and exchange of the second CST exon with the first CST exon, resulting in derived engineered cells expressing the first CST and GOI instead of the second CST and reporter gene, creating a combination system. 2. The combination system according to embodiment 1, wherein said first cell surface tag (CST) exon is different from said second CST. 3. The first component is a. a first RMCE element; b. 3' intron fragment, c.CST exon, d. a first transcription terminator; e. a second transcription terminator; f.GOI, g. Kozak sequence, h. Second RMCE element and wherein the CST exon and the first transcription terminator are encoded in the antisense direction from the GOI and associated transcription terminator and the Kozak sequence. 4. The first component is: a. First RMCE element—a 5′ RMCE element encoded within a “non-functional” non-coding 3′ intron fragment b. a 3' intron fragment containing a branchpoint sequence, a polypyrimidine tract, and a 3' splice acceptor site; c. an exon containing CST encoded by TEDV in the 5'→3' direction; d. a first transcription terminator sequence for CST encoded in the 5' to 3' direction; e. a second transcription terminator for the GOI encoded 3'→5'; f. a sequence encoding the GOI in the 3' to 5' direction; g. Kozak sequence, h.5'RMCE element, and the CST exon and the first transcription terminator are encoded in the antisense direction from the GOI and associated transcription terminator and the Kozak sequence.

[0037] 5. The first component is: a. a first RMCE element; b. 3' intron fragment, c.CST exon, d. a first transcription terminator; e. a second transcription terminator; f.GOI, g. Kozak sequence, h. a second RMCE element; and the CST exon and the first transcription terminator are encoded in the antisense direction from the GOI and associated transcription terminator and the Kozak sequence. 6. The second component is: a. transcriptional promoter element; b. Kozak sequence, c. transmembrane domain exon, d. introns, e. a first RMCE element; f.CST exon, g. a first transcription terminator; h. a second transcription terminator; i. a reporter gene, J. Kozak sequence, k. a second RMCE element; l. a second transcription promoter element; TERS, which includes 6. The combination system of any one of embodiments 1 to 5, wherein the transmembrane domain exon and the CST exon are encoded in an antisense direction from the reporter gene, and a first transcriptional promoter element drives transcription of the combination of the transmembrane domain and CST, and a second transcriptional promoter element drives transcription of the reporter gene. 7. The second component is: a. transcriptional promoter element; b. Kozak sequence, c. Type 2 membrane protein transmembrane domain exon, d. 5' intron splice donor site, e. a 5' RMCE element encoded by a "non-functional" non-coding 3' intron fragment that is an equivalent of and pairs with the 5' RMCE element of TEDV; f. a functional sequence of the 3' intron fragment that includes a branchpoint sequence, a polypyrimidine tract, and a 3' splice acceptor site; g. An exon containing a CST encoded by TERS in a 5' to 3' orientation (different from the CST encoded by TEDV); h. a transcription terminator sequence for CST encoded in the 5' to 3' direction; i. a transcription terminator sequence for the 3' to 5' sequence; j. a sequence encoding a selection gene in the 3' to 5' direction; k. Kozak sequence for efficient translation initiation of selected gene transcripts; l.3'RMCE element, m. 3' genomic elements responsible for regulating expression and tracking of CST transcripts; wherein the transmembrane domain exon and the CST exon are encoded in an antisense direction from the reporter gene, and wherein a first transcriptional promoter element drives transcription of the combination of the transmembrane domain and the CST, and a second transcriptional promoter element drives transcription of the reporter gene. 8. The second component is: a. transcriptional promoter element; b. Kozak sequence, c. transmembrane domain exon, d. introns, e. a first RMCE element; f.CST exon, g. a first transcription terminator; h. a second transcription terminator; i. a reporter gene, J. Kozak sequence, k. a second RMCE element; l. a second transcription promoter element; wherein the transmembrane domain exon and the CST exon are encoded in an antisense direction from the reporter gene, and wherein a first transcriptional promoter element drives transcription of the combination of the transmembrane domain and the CST, and a second transcriptional promoter element drives transcription of the reporter gene.

[0038] 9. A combination system described in any one of embodiments 1 to 8, wherein a first RMCE element of the TEDV is paired with a first RMCE element of the TERS, and a second RMCE element of the TEDV is paired with a second RMCE element of the TERS. 10. A combination system according to any one of embodiments 1 to 9, wherein each CST exon comprises a sequence encoding one or more molecular affinity tags, and the CST encoded by TEDV and the CST encoded by TERS are different. 11. The combination system according to any one of embodiments 1 to 10, wherein the engineered cell comprises a single TERS in the genome. 12. A method for producing a transgenic engineered cell that expresses a TEDV-encoded GOI from the TERS locus, comprising: a. Producing a TEDV encoding a GOI; b. delivering the TEDV to an engineered cell line containing a TERS paired with the TEDV, along with a recombinase enzyme that matches the RMCE element encoded by the TERS; c. contacting the cells with two or more affinity reagents specific for TEDV-encoded CST and TERS-encoded CST; d. To select cells with integrated GOI, select the derived engineered cells based on the attenuation of expression of the reporter gene and CST encoded by TERS and the increase of expression of CST encoded by TEDV. To do A method comprising: 13. The method of embodiment 12, wherein the affinity reagent used in step c is fluorescently labeled to detect the attenuation of expression of CST encoded by TERS and the increase of expression of CST encoded by TEDV, and allows for the partitioning and selection of cells based on said expression by fluorescence-activated cell sorting. 14. The method of embodiment 12, wherein the affinity reagent used in step c is immobilized on a substrate such that cells expressing TERS-encoded CST or cells expressing TEDV-encoded CST can be depleted or enriched in the target cell population using substrate affinity methods, such as magnetic-activated cell sorting. 15. Derivation of multiple engineered cells expressing a set of TEDV-encoded GOIs from a pool of TEDVs A method of making a. generating a library of two or more TEDVs, each encoding a unique GOI sequence and each having a unique CST encoded by the TEDV; b. delivering the TEDV library as a pool to an engineered cell line containing a TERS paired with the TEDV, along with a recombinase enzyme that matches the RMCE element encoded by the TERS; c. contacting the cells with three or more affinity reagents specific for multiple TEDV-encoded CSTs and TERS-encoded CSTs; d. selecting the engineered cells based on attenuation of expression of the reporter gene and TERS-encoded CST and increased expression of each of the unique TEDV-encoded CSTs; A method comprising:

[0039] 16. A method for tracing the cell lineage of engineered cells expressing a set of TEDV-encoded GOIs within a pool of cells produced by steps a and b of embodiment 15, comprising: a. contacting cells with two or more affinity reagents specific for multiple CSTs encoded by TEDV; b. The content of engineered cells was determined based on the expression of each unique CST encoded by TEDV. To analyze and A method comprising: 17.- Producing a library of two or more TEDVs, each encoding a unique GOI sequence and each having a unique CST encoded by the TEDV; - a method for tracing the lineage of derived engineered cells expressing a set of TEDV-encoded GOIs in a pool of cells produced by delivering the TEDV library as a pool into an engineered cell line containing a TERS paired with the TEDV, together with a recombinase enzyme that matches the RMCE element encoded by the TERS, comprising: a. contacting cells with two or more affinity reagents specific for multiple CSTs encoded by TEDV; b. Analyzing the content of the engineered cells based on the expression of each unique CST encoded by TEDV; A method comprising: 18. A tag exchange donor vector (TEDV) encoding a cell surface tag (CST) exon flanked by 3′ intron fragments and a gene of interest (GOI) in the antisense orientation. 19.a. First RMCE element; b. 3' intron fragment, c.CST exon, d. a first transcription terminator; e. a second transcription terminator; f.GOI, g. Kozak sequence, h. Second RMCE element 20. The tag exchange donor vector (TEDV) of embodiment 18, comprising: a CST exon and a first transcription terminator encoded in the antisense direction from the GOI and associated transcription terminator and Kozak sequence. 20. An engineered cell comprising a tag exchange receiver site (TERS) in its genome, the TERS encodes a cell surface tag (CST) exon and encodes a reporter gene in an antisense orientation, the CST exon being connected to an exon encoding a transmembrane domain by a full-length intron sequence; Here, a recombinase-mediated cassette exchange (RMCE) element is contained in the TERS, and RMCE between the TERS and a tag exchange donor vector (TEDV) results in the exchange of a gene of interest (GOI) encoded by the TEDV with a reporter element in engineered cells.

[0040] 21. The TERS: a. transcriptional promoter element; b. Kozak sequence, c. transmembrane domain exon, d. introns, e. a first RMCE element; f.CST exon, g. a first transcription terminator; h. a second transcription terminator; i. a reporter gene, J. Kozak sequence k. a second RMCE element; l. Second transcription promoter element Including, 21. The engineered cell of embodiment 20, wherein the transmembrane domain exon and the CST exon are encoded in an antisense direction from a reporter gene, and wherein a first transcriptional promoter element drives transcription of the combination of the transmembrane domain and CST, and a second transcriptional promoter element drives transcription of the reporter gene. [Example]

[0041] Materials and Methods Incorporation of TERS into engineered cell lines Electroporation was used to deliver the necessary DNA constructs to generate engineered cells with a single TERS site integrated into the AAVS1 site by homologous recombination. 4 x 10 per reaction 6 Cells were pulsed in the Gene Pulser Xcell with the following settings: square wave 285V, pulse length 12.5ms, 2 pulses spaced 1s apart. TM Electroporation was performed in 500 μl of RPMI 1640 containing Glutamax-I (Life Technologies) using a Bio-Rad. The DNA concentrations used were 15 μg / μl for the TERS integration vector (V9.F.5), 10 μg / ml for the Cas9-P2A-GFP coding plasmid (V1.A.8), and 7.5 μg / ml for the vector encoding the gRNA targeting the AAVS1 site (V2.J.6), the integration site (Table 3). After electroporation, cells were incubated in RPMI 1640 medium containing Glutamax-I + 10% FBS for 2 days (37°C, 5% CO2) before analysis.

[0042] Sorting of polyclonal GFP-expressing transfectant cells Cells electroporated with a plasmid encoding Cas9-P2A-GFP (V1.A.8) or a GFP selectable marker (V1.A.4) were analyzed by FACSJAzz TM Transient GFP expression was sorted using a cell sorter (BD Biosciences). Cells were washed and resuspended in an appropriate volume of DPBS before sorting in RPMI 1640 containing Glutamax-I, 20% HI-FBS, and Anti-Anti 100X (Life Technologies).

[0043] Selection of monoclonal cells stably expressing TERS Single cells constitutively expressing the Myc epitope CST and RFP selection gene expressed from the TERS receptor cassette were obtained using FACS. To detect Myc CST, cells were stained with an anti-Myc antibody (anti-c-Myc-Alexa 647, SantaCruz) and then sorted. Cells were washed with DPBS and then sorted in RPMI 1640 containing Glutamax-I, 20% HI-FBS, and Anti-Anti 100X (Life Technologies).

[0044] [Table 1]

[0045] GFP and RFP fluorescence were measured using the filter sets listed in Table 4. TM Detection was performed by FACS (BD Biosciences). Single cells expressing Myc and RFP were sorted into 96-well plates containing 200 ul of growth medium, and monoclonal collections were grown.

[0046] [Table 2]

[0047] Phenotypic screening of monoclonal populations A sample of 20,000 cells from the grown monoclonal collection was transferred to a microtiter plate for analysis, and the cells were resuspended in 250 ul of DPBS 1X (Life Technologies) and incubated in LRS Fortessa TM The monoclonal population (ACL-1163) was screened for the presence of the Myc epitopes CST and RFP. Myc expression was detected using an anti-Myc antibody labeled with Alexa Fluor 647 fluorophore. The staining solution was prepared using the recommended antibody volume diluted in 100 μl of staining buffer (DPBS + 2% FBS). Cells were incubated at 4°C for 1 hour and then washed twice with 500 μl of staining solution before analysis.

[0048] Genotypic screening of monoclones - confirmation of integration at the correct genomic location ACL-1163 cells were maintained in normal growth medium of RPMI 1640 containing Glutamax-I + 10% HI-FBS. 6 The cell confluency was monitored daily until it reached 5 × 10 6 Cells were extracted using a QIAamp DNA mini kit (Qiagen). The remaining cells were further expanded and cryopreserved at a density of 3 x 10 cells / ml in 70% growth medium + 20% HI-FBS + 10% DMSO. The ACL-1163 monoclone was screened and evaluated at the molecular level. This was performed by PCR using Q5® Hot Start High Fidelity DNA Polymerase (NEB) in a 20 μl reaction, using the components and reaction conditions listed in Tables 5 and 6, respectively. To determine whether the TERS integration cassette had integrated into the AACS1 locus, primers 15.F.9 and 19.E.7 were used (Table 7). These primers target the region preceding the left homologous arm and the transmembrane domain, respectively. Correct left homologous arm recombination was indicated by a 2.1 kb amplicon. First, a PCR master mix was prepared using all components (Q5® Reaction Buffer, dNTPs, Hot Start Q5® DNA Polymerase, primers Fwd and Rev, 100 ng of DNA template, and HO). PCR reactions were performed using a C1000 Touch TM The PCR products were run on a 1% agarose gel in 1x TAE buffer using a PowerPac Basic (BioRad) and stained with 1:10,000 diluted Sybersafe and purified with Fusion SL (Vilber Lourmat). was used for the analysis.

[0049] [Table 3]

[0050] [Table 4]

[0051] [Table 5]

[0052] Identification of gene copy number The DNA of selected monoclones was evaluated for the number of TERS cassettes integrated into the cell genome. To achieve this, droplet digital PCR (ddPCR) was performed using primers and probes specific for the TERS cassette and the reference gene (TRAC) (Table 8). The TERS-specific probe was conjugated with FAM, and the reference gene-specific probe was conjugated with HEX. Based on the integrated copy number, ACL-1163 cells were considered diploid for the reference gene (TRAC). Prior to ddPCR, DNA was digested with MfeI (NEB) to isolate tandem integrations. The reaction setup and cycling conditions were the same as for ddPCR for the probe. TM QX200 Supermix (without dUTP) (Bio-Rad) was prepared according to the protocol. TM Droplet Reader and Droplet Generator and C1000 Touch TM A deep-well thermal cycler (Bio-Rad) was used. Data were obtained from QuantaSoft. TM The software was used to acquire and detect FAM using Ch1 and HEX using Ch2.

[0053] [Table 6]

[0054] Flp-mediated integration of GOI sequences in derivative engineered cell lines Electroporation was used to deliver the required DNA constructs to facilitate Flp recombinase-mediated tag exchange. 4 × 10 per reaction 6 Cells were pulsed in a Gene Pulser Xcell with the following settings: square wave 285V, pulse length 12.5 ms, 2 pulses separated by 1 second. TMElectroporation was performed in 500 μl of RPMI 1640 containing Glutamax-I (Life Technologies) using a Bio-Rad. The DNA concentrations used were 7.5 μg / ml for the TEDV vector (V9.F.5), 10 μg / ml for the FLPO-encoding plasmid (V12.A.8), and 7.5 μg / ml for the vector encoding GFP (V1.A.4) to trace DNA delivery (Table 3). After electroporation, cells were incubated in RPMI 1640 medium containing Glutamax-I + 10% FBS for 2 days (37°C, 5% CO2) before analysis and cell sorting for GFP-positive cells.

[0055] Phenotyping for tag exchange To determine whether Flp recombinase-mediated tag exchange had occurred, cells were stained for surface expression of Myc and SBP, and RFP fluorescence intensity was measured. Cells were harvested 7-10 days after electroporation and surface stained for SBP and Myc using the following antibodies (anti-SBP-Alexa647 and anti-c-Myc-AlexaPE, SantaCruz). GFP and RFP fluorescence were detected using Influx Fluorescence Imaging (IFVI) with the filter sets listed in Table 4. TM (BD Biosciences) and detected by FACS. Single cells expressing SBP but not Myc or RFP were sorted and transferred to 96-well plates containing 200 ul of growth medium to grow monoclonal collections. Phenotyping of monoclones was performed 20–24 days after single-cell sorting. For flow cytometry analysis, cells were removed from the wells and 300 μl of RPMI was added per tube. Cells were centrifuged at 400 g for 3 minutes at 4°C, the supernatant was aspirated, and the cell pellet was resuspended in 25 μl of staining mix (staining mix: anti-SBP-Alexa647 and anti-c-Myc-AlexaPE) or RPMI (unstained control) and incubated at 4°C for 30 minutes. Cells were washed twice with staining buffer (SB) (DBPS + 2% FBS) and centrifuged at 400 g for 3 minutes. Cells were resuspended in 200 μl of SB, transferred to a 96-well plate, and data were acquired using an LSRFortessa. Analysis was performed using FlowJo.

[0056] Confirmation of GOI expression After growth and harvesting, cells were lysed in 150 mM NaCl, 50 mM Tris pH 8, 1% CHAPS, 5 mM imidazole, 1 mM PMSF, and 1x protease and phosphatase inhibitors (Thermo) for 20 min at 4°C on a rotor. Lysates were cleared by centrifugation at 17,000 g for 10 min at 4°C and subjected to sodium dodecyl sulfate (SDS) gel electrophoresis on a 10% acrylamide precast gel (Biorad) at 140 V for 1 h. Gels were turboblotted onto PVDF membranes (Biorad), which were then blocked for 15 min in SeaBlock 1x (Thermo) in Tris-buffered saline / tween 20 (TBST) 1x and incubated with mouse anti-flag antibody (Sigma) for 2 h at room temperature. The membrane was washed to remove unbound primary antibody for 3 × 5 min in TBST 1× and then incubated with anti-mouse horseradish peroxidase (HRP)-conjugated goat antibody for 1 h at room temperature. Finally, the membrane was washed to remove unbound secondary antibody for 3 × 5 min in TBST 1×, and the HRP signal was developed with ECL substrate (Biorad) and acquired on a Fusion SL Vilber system.

[0057] Confirmation of GOI genome integration Monoclonal cell lines expressing cell surface tag 2 (SBP) were evaluated at the molecular level for cointegration of the GOI coding sequence. This was performed by PCR using Q5® Hot Start High Fidelity DNA Polymerase (NEB) in a 30 μl reaction, with the components and reaction conditions listed in Tables 9 and 10, respectively. Primers 12.G.5 and 21.G.8 were used to determine whether the GOI had integrated into the genome (Table 7). These primers target the 3'UTR region of the GOI. First, a PCR master mix was prepared with all components (Q5® Reaction Buffer, dNTPs, Hot Start Q5® DNA Polymerase, primers Fwd and Rev, 100 ng of DNA template, and HO). PCR reactions were performed using a C1000 Touch TM The PCR was performed using a thermal cycler (Bio-Rad). PCR products were run on a 1% agarose gel in 1x TAE buffer using a PowerPac Basic (Bio-Rad), stained with 1:10,000 dilution of Sybersafe, and analyzed using Fusion SL (Vilber Lourmat). Sanger sequencing confirmed that the band of the correct size encoded the sequence of the GOI 3'UTR.

[0058] [Table 7]

[0059] [Table 8]

[0060] MACS enrichment / depletion of surface tag-expressing cells The manufacturer's MACS protocol for enrichment / depletion was generally followed (Miltenyi Biotec, #130-047-101, IM0001377.PDF).

[0061] Sample preparation Cells were harvested and washed once by centrifugation (300 × g, 3 min at 4°C) in staining buffer-M (SB-M: cold Dulbecco's phosphate-buffered saline (DPBS), 2% FBS, 2 mM EDTA). Cells resuspended in SB-M were filtered through a 40 μm cell strainer to obtain a single-cell suspension. Cells were washed with 3 ml of cold SB-M, and the cell pellet was collected. Samples were incubated for 30 min at 4°C with 80 μl of staining solution containing the appropriate antibody or dead cell removal reagent per 10 million cells. Cells were washed twice with 3 ml SB-M buffer and pelleted.

[0062] Magnetic bead labeling Pelleted cells were labeled with magnetic beads by resuspending the cells in 160 μl of SB-M and adding anti-mouse IgG1 MACS microbeads (Miltenyi) (40 μl MACS microbeads were added per 10 million cells). After a 20 minute incubation at 4°C, the cells were washed with 3 ml SB-M and finally resuspended in 500 μl SB-M.

[0063] Magnetic separation The LS column (Miltenyi) was placed in the magnetic field of an appropriate MACS separator (Miltenyi) and rinsed with 3 ml SB-M. The MACS pre-fraction was also collected. The cell suspension was added to the column, and the flow-through fraction containing unlabeled cells was collected in a 15 ml conical tube (referred to as the flow-through fraction). The column was removed from the separator and placed in an appropriate collection tube. 5 ml SB-M was added to the column containing the magnetically labeled cells. Pressure was applied using the supplied plunger until the plunger reached the bottom of the column. The magnetically labeled cells were eluted from the column (referred to as the bound fraction), and this fraction was used for downstream applications.

[0064] Example 1 - Incorporation of TERS into engineered cell lines This example describes the stable integration of TERS into a cell line to generate the engineered cell line monoclonal ACL-1163, which contains a single TERS in its genome. In this example, the TERS, represented as SEQ ID NO: 1, was composed of the following selected genetic elements encoding two genes: the first gene, encoded in the sense direction, contains an EF1a promoter upstream of an ORF spanning two exons. The first exon encodes a transmembrane type II protein domain (TD), and the second exon encodes a Myc epitope tag. The intron between the two exons is derived from the human GAPDH gene and was modified to encode a first heterologous-specific FRT site (FRT) between the 5' intron splice donor site and the intron branch point sequence. The 3' end of the ORF encodes the SV40 polyadenylation signal terminator. The second gene, encoded in the antisense direction, contains an EF1a promoter upstream of an ORF encoding the fluorescent reporter RFP. The region between the Kozak sequence and the promoter encodes a second heterologous-specific FRT site (F3). The 3' end of the RFP ORF encodes the bGHpA polyadenylation signal terminator. A plasmid was constructed to facilitate stable genomic integration of TERS into the AAVS1 genomic safe harbor locus. In this plasmid, the TERS DNA element was flanked by left and right homologous arms of AAVS1. Each arm consisted of >500 bp of sequence homologous to the AAVS1 genomic locus. Stable integration of TERS was achieved by the process of homologous recombination (HDR) at the AAVS1 genomic safe harbor locus.

[0065] The ACL-128 cell line was transfected with a plasmid encoding the TERS gene element flanked by the right and left homologous arms of AAVS1, a plasmid encoding an optimal gRNA targeting the AAVS1 locus, and a plasmid encoding Cas9-P2A-GFP. After 2 days, cells positive for Cas9-P2A-GFP plasmid uptake were FACS-sorted based on GFP fluorescence. GFP-sorted cells were further expanded for >7 days. TERS-transfected cells were stained with an anti-Myc antibody and analyzed by flow cytometry for the presence of RFP and the Myc epitope CST (Figure 6a). Cells with TERS integration into their genome showed increased RFP and Myc signals. These cells were then sorted, expanded, and collected into monoclonal collections. A representative monoclone, ACL-1163, shown in Figure 6b, exhibited stable and robust RFP and Myc surface expression. To confirm integration of the genomic receiver cassette into the targeted AAVS1 site, genomic DNA was extracted from the selected ACL-1163 cell line and PCR reactions were performed using primers internal to the TERS receiver cassette and primers specific to the AAVS1 locus (15.F.9 and 19.E.7, see Figure 7). PCR amplicons of the expected size were detected (data not shown). Furthermore, ddPCR using primers and probes (forward primer 1.I.7, reverse primer 1.I.8, probe 1.I.9, see Table 8) confirmed that only a single copy of the TERS receiver cassette had been integrated (data not shown). The resulting engineered cell line, ACL-1163, contained a single copy of TERS designed for RMCE with the appropriate TEDV paired.

[0066] Example 2 - RMCE with TEDV with tag exchange and GOI delivery for generation of derived engineered cells This example demonstrates that CSTE, an RMCE-driven reaction between TEDV-encoded sequences and TERS, results in the exchange of CST on the cell surface, thereby reporting the exchange construct incorporating the TEDV-encoded sequence and GOI. In this example, ACL-1163, described above, was used as the target-engineered cell line. The TEDV in this example encodes, in the sense orientation, a first heterologous-specific FRT site (FRT); a 3' intron fragment containing a branchpoint sequence, a polypyrimidine tract, and a 3' splice acceptor site; and an exon encoding a streptavidin-binding peptide (SBP) and the SV40 polyadenylation signal terminator. Three distinct GOIs derived from respiratory syncytial virus (RSV) were encoded in the antisense orientation in separate TEDVs, with each GOI ORF located between a second heterologous-specific FRT site (F3) and the 3' bGHpA polyadenylation signal terminator. The sequences in SEQ ID NOs: 3 to 5 represent the three independent TEDV constructs used, encoding the SBP epitope, CST, and GOI.

[0067] In this example, the engineered cell line ACL-1163 constructed in Example 1 was electroporated with an expression vector (FLPO, V4.1.8, Table 3, SEQ ID NO: 2) encoding TEDV (selected from the sequences of SEQ ID NO: 3 to SEQ ID NO: 5) and the RMCE recombinase enzyme. The cells were incubated for 7 to 10 days to allow integration couples to develop, and then stained with anti-Myc and anti-SBP antibodies and analyzed by flow cytometry for RFP, Myc, and SBP reporter signals. Cells showing a decrease in RFP and Myc signals and an increase in SBP signal, indicative of "tag exchange," were selected and expanded to form a collection of monoclones. Characterization of a representative monoclone, ACL-3426, is shown in Figures 7a and 7b. To confirm the success of surface tag exchange, monoclone ACL-3426 was stained with antibodies against Myc and SBP and analyzed by flow cytometry for loss of RFP and Myc surface expression (Figure 7a) and gain of SBP signal (Figure 7b). Indeed, cells that underwent CSTE showed a signal for SBP but no signal for Myc or RFP, indicating successful tag exchange. Parallel analysis of parental cells showed that high RFP and Myc signals (Figure 7a, right panel) and low SBP signals (Figure 7b, right panel) persisted.

[0068] To verify that the GOI ORFs were integrated and expressed after CSTE, three monoclones from independent experiments using each of the above TEDVs encoding different RSV-1 GOIs were evaluated by immunoblotting (Fig. 7c). The GOI ORFs provided by the TEDVs were the three RSV-1 genes P, N, and M2 (long), each encoding a Flag tag. By performing CSTE, cells were transfected with a construct encoding flp recombinase and either a TEDV encoding the SBP epitope CST and the RSV-1 P gene (resulting in a cell line monoclone: ​​ACL-3374); a TEDV encoding the SBP epitope CST and the RSV-1 N gene (resulting in a cell line monoclone: ​​ACL-3386); or a TEDV encoding the SBP epitope CST and the RSV-1 M2 long gene (resulting in a cell line monoclone: ​​ACL-3433). Proteins were extracted from the monoclones, and the samples were immunoblotted with mouse anti-Flag primary antibody followed by incubation with anti-mouse horseradish peroxidase (HRP)-conjugated goat antibody. The HRP signal was developed using ECL substrate. Successful CSTE was indicated by the presence of a single positive band for Flag at the expected molecular weight for each GOI (Figure 7c, lanes 2-4). Parental cells were analyzed in parallel, but no Flag-tag signal was present (Figure 7c, lane 5). In summary, this example demonstrates that cell surface tag exchange can be used to conditionally report the presence of the initial TERS construct and the exchanged construct in which TEDV-encoded sequences have been integrated by RMCE, allowing for reporting of GOI integration and expression independently of detection of the GOI itself.

[0069] Example 3 - Enrichment of Tag2 (SBP) or depletion of Tag1 (Myc) from mixed engineered cell populations after tag exchange by magnetic activated cell sorting (MACS) This example demonstrates that surface tag technology can be used for Tag2 (SBP) enrichment or Tag1 (Myc) depletion by MACS. Furthermore, the presence of Tag2 (SBP) can be used to monitor gene of interest (GOI) integration after tag exchange. A starting mixed population of two engineered monoclonal cell lines, APL-3015 and APL-4535, was used. The cell population was mixed, with 90% Myc-positive APL-3015 cells and 10% SBP-positive APL-4535 cells. The tag exchange receiver site (TERS) in APL-4535 cells encoded the SBP epitope CST and a full-length FLAG-tagged GOI encoding an intracellular protein, while the TERS in APL-3015 cells encoded the Myc epitope CST and an RFP selection gene. Magnetic-activated cell sorting (MACS) was used to enrich for SBP-positive APL-4535 cells from the mixed population. In a separate experiment, MACS was used to deplete Myc-positive APL-3015 cells from a mixed population.

[0070] In the first case, a mixed cell population was labeled with anti-SBP-Alexa 488 fluorophore and then incubated with anti-mouse IgG iron beads. SBP-labeled cells were enriched using MACS and counterstained with anti-c-Myc-Alexa 405 fluorophore. Figure 8a shows the ratios of SBP- and Myc-positive cells at three experimental steps: 1) pre-MACS to determine the starting ratio of SBP- and Myc-positive cells in the mixed cell population; 2) flow-through to assess the ratio of SBP- and Myc-positive cells not captured by the MACS column when it was placed in a magnetic field; and 3) bound fraction to assess the ratio of SBP- and Myc-positive cells captured by the MACS column. All three fractions were counterstained with anti-c-Myc-Alexa 405 fluorophore, and data were acquired on a BD Influx instrument. Figure 8a shows successful enrichment of SBP-positive cells due to the absence of SBP-positive cells in the flow-through fraction (<0.01), while after SBP-targeted enrichment, 95% of the bound cells were SBP-positive.

[0071] To demonstrate that surface tagging technology can be used to deplete Tag1 (Myc)-expressing base cell lines from mixed cell populations by MACS, all cells were labeled with anti-c-Myc-Alexa 405 fluorophore and incubated with anti-mouse IgG iron beads. Labeled cells were depleted using MACS, and the indicated fractions were collected. All three fractions were counterstained with anti-SBP-Alexa 488, and data were acquired on a BD Influx instrument. Successful depletion of Myc-positive cells was evidenced by a reduction in the number of Myc-positive cells (25%) and an increase in the number of SBP-positive cells (75%) in the flow-through fraction. To further demonstrate that the presence of Tag2 (SBP) can be used as a reporter to monitor GOI integration after tag exchange, a total of 546 SBP-positive monoclones were individually evaluated for their ability to encode a GOI linked to SBP. The chart in Figure 8c shows that 96.52% of SBP-positive cells encoded a GOI, while 3.48% of SBP-positive cells did not express a GOI. These results demonstrate that the surface tagging technique is suitable for enriching for Tag2(SBP)-expressing monoclones or depleting the unmodified base cell line (Tag1(Myc)-expressing monoclones) from a mixed cell population after tag exchange. Furthermore, because the presence of Tag2(SBP) correlates with GOI integration after tag exchange, the presence of Tag2(SBP) on the cell surface can be used as an indicator of successful GOI delivery to the TERS in engineered cell lines.

[0072] Example 4 - Construction and operation of a barcode-based cell surface tag exchange (CSTE) system This example provides a schematic overview of the concept of barcoding engineered cells expressing a GOI using the CSTE system. Figure 9 is a schematic diagram of the use of the CSTE system to barcode cells expressing a GOI. Panels a and b show the system components, including a pool of tag-exchange donor vectors (TEDVs) and engineered cells containing tag-exchange receiver sites (TERS), respectively.

[0073] Each TEDV encodes an RMCE element at the 5' and 3' ends of the construct, paired with the RMCE site contained in the TERS. The RMCE element at the 5' end of the TEDV construct is encoded within the 3' intron fragment sequence, and immediately 3' to this RMCE element is an intronic 3' element containing the branchpoint sequence, polypyrimidine tract, and splice acceptor site. Thus, RMCE is contained within a "non-functional" non-coding intron sequence, and the 3' intron fragment contained in the TEDV lacks a 5' splice donor site. Immediately 3' to the splice acceptor site, the TEDV encodes a 3' exon of a cell surface tag (CST). This exon encodes a portion of CST containing a unique molecular binding motif. While this CST sequence is encoded in the 5'-to-3' direction, the TEDV also encodes a gene of interest (GOI) to be integrated into the TERS, encoded in the 3'-to-5' direction.

[0074] Each CST in this example is composed of two epitopes selected from three unique epitopes (A, B, and C) (six unique combinations are possible). Because the location of each epitope is difficult to distinguish using current technology, AB is essentially equivalent to BA. In this example, the AX combination was assigned to a GOI family with three variants (GOIs ai, a-ii, and a-iii), and the BX combination was assigned to a second GOI family with three variants (GOIs bi, b-ii, and b-iii). The individual TEDVs in this example were pooled (Figure 9a). The central portion of the TERS gene in engineered cells encodes elements that are similar in structure but distinct from those in TEDV. Specifically, TERS encodes a CST exon distinct from the TEDV-encoded CST, located between the RMCE element paired with that in TEDV. This CST exon contains a splice acceptor site and associated 3' intron sequence immediately 5' from the CST. Similarly, a selected gene, similar to the GOI in TEDV, is encoded in the antisense orientation within TERS. The 5' end of the construct contains a promoter sequence driving CST transcription. 3' to this promoter sequence, a transmembrane domain (TD) exon is encoded, with a 5' intron sequence immediately 3' from that. This means that the TD and CST exons, encoded in frame with the RMCE element-containing intron, are generated as a continuous transcript, which is then spliced ​​to join the exons into a single coding mRNA. The TD-CST product encoded by TERS is expressed on the cell surface. At the 3' end of the TERS construct there is another promoter element that drives transcription of the selection gene in the 3' to 5' direction, resulting in expression of the selection gene (Figure 9b).

[0075] Introduction of the TEDV pool into a TERS-containing engineered cell population, along with an appropriate expression construct for a recombinase specific for the paired RMCE element in TEDV / TERS, results in RMCE, generating a pool of derived engineered cells expressing the GOI (Figure 9c). The TERS-encoded element is replaced by a TEDV-encoded element. The derived cell population thus expresses the TEDV-encoded CST on the cell surface as a TD-CST product, originally containing the TERS-encoded TD, and expresses the GOI. In summary, RMCE between a TEDV and TERS pool generates a derived engineered cell pool that loses expression of the selected gene and the CST originally encoded by TERS, while each cell in the pool acquires expression of one of the TEDV-encoded CST members of the TEDV pool and the GOI. The GOI-expressing engineered cell pool can be further analyzed / isolated into individual members by FACS, for example, based on expression of unique CST barcodes (Figure 9d). This can be achieved as a bulk population of the desired barcode or by individual cell isolation using single-cell sorting. Alternatively, analysis of the pool can be performed by FACS without sorting, using the barcodes as a means to identify populations of interest within the digital dataset, along with secondary analysis of cellular function, for example, correlating variant GOI expression or cellular function.

[0076] To prove the concept that CST can be composed of multiple epitopes, ACL-1 and ACL-5 cells were transfected with either a plasmid encoding CST or a control plasmid lacking CST (Figure 9e). This CST contained three unique epitopes: FLAG, MYC, and HA (as indicated by the sequence of SEQ ID NO: 20). Cells were transfected using chemical transfection (ACL-5) or electroporation (ACL-1) using standard methods known to those skilled in the art. After 48 hours, cells were harvested and stained with fluorophore-conjugated cognate antibodies: anti-FLAG-PE, anti-MYC-AF647, and anti-HA-AF488. Cells were analyzed by flow cytometry. Viable cells were gated by forward scatter (FSC) and side scatter (SSC). The mean fluorescence intensity (MFI) of viable cells was determined for each of the three epitopes, and the percentage of viable cells expressing each epitope was determined. All three epitopes were detected at higher rates and intensities in cells transfected with the barcoded CST-encoding plasmid compared to empty vector-transfected samples, demonstrating the ability of CST to be composed of multiple epitopes. These results demonstrate that surface tag technology is suitable for barcoding cell lines, as CSTs can consist of multiple epitopes.

[0077] List of abbreviations AAVS1 adeno-associated virus integration site 1 APC antigen presenting cells Cas9 CRISPR-related gene 9 CMV cytomegalovirus cre Cre recombinase CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CST Cell Surface Tags CSTE Cell Surface Tag Exchange DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DPBS Dulbecco's Phosphate Buffered Saline DSB double-strand break dUTP deoxyuridine triphosphate EDTA Ethylenediaminetetraacetic acid EF1 alpha elongation factor alpha (for eukaryotic translation) FACS Fluorescence-activated cell sorting FAM Fluorescein amidite FBS Fetal Bovine Serum FLP flippase FRT flippase recognition target GFP Green Fluorescent Protein GOI Gene of Interest gRNA guide ribonucleic acid HDR Homologous Recombination HLA human leukocyte antigen IRES internal ribosome entry site MACS Magnetic Activated Cell Sorting NEB New England BioLabs NHEJ non-homologous end joining

[0078] ORF Open Reading Frame PCR polymerase chain reaction RFP Red Fluorescent Protein RMCE Recombinase-Mediated Cassette Exchange RPMI Roswell Park Memorial Institute RSV respiratory syncytial virus RT reverse transcription RNA ribonucleic acid SBP Streptavidin-binding peptide SSR site-specific recombinase SV40 simian virus 40 SV40pA simian virus 40 poly(A) TAA tumor-associated antigen TALEN transcription activator-like effector nucleases TAE Tris Acetate-EDTA T cell T lymphocyte TCR T cell receptor TCS target coding sequence TD transmembrane domain TEDV tag exchange donor vector TERS tag exchange receiver section rRNA ribosomal RNA tRNA transfer RNA UTR untranslated region ZNF zinc finger nuclease

[0079] List of definitions Amplicon: A piece of DNA or RNA that is the source and / or product of artificial amplification using various methods, including PCR. Antibody: A two-chain affinity molecule expressed by specialized cells of the immune system called B cells. B cells express a very large and highly diverse repertoire of antibodies that do not normally bind to self-proteins but can bind to and neutralize pathogens or toxins that may pose a threat to the host. Natural or engineered antibodies may be used as affinity reagents. Nutritional requirement: A mutant organism (especially a bacterium or fungus) that requires a specific additional nutrient not required by the normal strain. cis-acting element: a non-coding DNA region that regulates the transcription of nearby ORFs. CST: A co-integrated cell surface tag that allows reporting of the integrated gene of interest. CSTE system: A system that operates as a donor / receiver pair, in which a tag exchange donor vector acts to deliver a gene sequence of interest and a cell surface tag exon to a paired tag exchange receiver site contained within the genome of an engineered cell line. Derived engineered cells: engineered cells that have been further genetically modified to replace the CST and incorporate the GOI.

[0080] DNA: Deoxyribonucleic acid, the chemical name for the molecules that make up the genetic material that codes for genes and proteins. Engineered cell: A cell whose genome has been manipulated through genetic modification. Epitope: The region of an antibody target to which an antibody or other affinity reagent binds. Eukaryotic conditional regulatory element: A DNA sequence capable of influencing the activity of a promoter that can be induced or repressed under defined conditions. Eukaryotic promoter: A DNA sequence that encodes an RNA polymerase binding site and response elements. The promoter plays a major role in determining where and when a gene of interest is expressed, as the sequence of the promoter region controls the binding of RNA polymerase and transcription factors. Eukaryotic terminator / signal terminator: DNA sequence recognized by a protein factor that binds RNA polymerase II and triggers the transcription termination process. It also encodes a poly(A) signal.

[0081] FACS / Flow Cytometry: Fluorescence Activated Cell Sorting. Flow cytometry is a technique that allows individual cells to be analyzed en masse for the expression of specific cell surface and intracellular markers. Variations on cell sorting techniques allow cells bearing a defined set of markers to be collected for further analysis. Flippase: A recombinase (flippase, Flp) derived from the 2 μm plasmid of the baker's yeast Saccharomyces cerevisiae. Fluorescent (protein) marker: A molecule that has specific quenching and emission properties and can be detected by microscopy, FACS, and related techniques. Genetic cis-acting elements: present on the same DNA molecule as the gene they regulate. Trans-regulatory elements, on the other hand, can regulate genes distant from the gene they transcribe. Cis-regulatory elements are often binding sites for one or more trans-acting factors. Genetic barcoding: DNA barcoding is a classification method that uses short genetic markers in an organism's DNA to identify it as belonging to a particular species. GOI: Gene of interest is defined as any nucleic acid coding or non-coding sequence of interest.

[0082] heterospecific recombinase site: a DNA sequence recognized by a recombinase enzyme that promotes the crossover of two DNA molecules. Homologous arm: A stretch of DNA that has near-identical sequence identity with a complementary homologous arm, thus facilitating the exchange of two DNA molecules by the cellular process of homology-directed repair. Insulator: a DNA sequence that prevents genes from being affected by the activation or repression of nearby genes. Insulators also prevent the spread of heterochromatin from silenced genes to actively transcribed genes. Integration: The physical ligation of a DNA sequence into a cell's chromosome. Internal ribosome entry site (IRES): A DNA sequence that encodes an RNA element that, when transcribed, allows translation initiation in a cap-independent manner. Intron: A non-coding section of an RNA transcript or the DNA that codes for it that is spliced ​​out before the RNA molecule is translated into a protein. Intron branchpoint sequence: The branchpoint sequence nucleotides that initiate a nucleophilic attack on the 5' splice donor site. The free end of the upstream intron then initiates a second nucleophilic attack on the 3' splice acceptor site, releasing the intron as an RNA lariat and covalently linking the two exons.

[0083] K: Nucleotide code for Keto (K=G or T). Kozak sequence: a short sequence required for efficient translation initiation. M: Nucleotide code for amino (aMino) (M=A or C). MACS: Magnetically Activated Cell Sorting: A cell isolation technique in which cells are labeled with affinity molecules containing magnetic particles for separation using a magnetic field. Matched: Two components are matched when they encode genetic elements that direct and limit the interaction between complementary components. Monoclonal cell line: A defined population of cells that arises from a single ancestral cell by repeated cell replication. N: Nucleotide code representing any (aNy) nucleotide (N=A, T, C, or G). Native: An entity that occurs naturally to a cell. Negative selectable marker: A selectable marker that allows for negative selection of a vector and / or a host organism that harbors a marker-carrying vector. Non-coding gene: a DNA sequence that does not encode a protein and is transcribed into a functional non-coding RNA molecule. Origin of replication: A specific sequence within a vector, plasmid, or genome where replication begins.

[0084] ORF: Open Reading Frame. A stretch of genetic material that encodes the translation frame for protein (polypeptide) synthesis by ribosomes. Overhang: A single-stranded sequence at the end of a double-stranded nucleic acid molecule, often called a sticky or cohesive end. PCR: Polymerase chain reaction in which specific target DNA molecules are exponentially amplified. Peptide: A short stretch of amino acids, typically 6 to 30 amino acids in length. Phenotypic analysis: analysis of the observable characteristics of cells. Plasmid: A genetic construct capable of replicating independently of a chromosome, typically a small circular strand of DNA in the cytoplasm of bacteria or protozoa. Polypeptide: a protein consisting of peptide stretches that form a three-dimensional structure. Polypyrimidine motif: A pyrimidine-rich motif (CAG) located upstream of the 3' end of the intron. n T n )motif. Positive selectable marker: A selectable marker that allows for positive selection of vectors and / or host organisms that carry the marker-carrying vector.

[0085] Primer: A short DNA sequence that allows for specific recognition of a target DNA sequence, for example during PCR. Promoter: A regulatory DNA element that controls the initiation of gene expression. Recombinase: An enzyme that mediates genetic recombination and catalyzes RMCE. Reporter element: A genetic element that mediates a signal that is reported in an organism or vector that harbors it. Can be used as a positive or negative selectable marker. Restriction enzyme cleavage sequence: A gene sequence that is cleaved by a restriction enzyme, which may be unique or unique to the recognition sequence of the restriction enzyme. Restriction enzyme recognition sequence: A genetic sequence that is recognized by a restriction enzyme and to which the enzyme binds. RMCE: Recombinase-Mediated Cassette Exchange. The exchange of genetic material at genomic receiver sites catalyzed by a recombinase. Splice acceptor site: The DNA sequence at the 3' end of an intron. Splice donor site: DNA sequence at the 5' end of an intron. Suicide gene: A gene that mediates cell death in the host organism that harbors it. Can be used as a positive or negative selectable marker. Synthetic: An artificially created entity.

[0086] TEDV: A tag exchange donor vector paired with a tag exchange receiver site contained within the genome of an engineered cell, which is used to deliver a gene of interest and a cell surface tag exon. TERS: tag exchange receiver site paired with TEDV contained within the genome of the engineered cell line. Type II transmembrane domain: a single, non-cleavable transmembrane stretch consisting of hydrophobic residues near the N-terminus that acts as a combined signal / anchor sequence, with the N-terminal part inside the membrane and the C-terminal part exposed to the extracellular or ER lumen. Vector: A vector is a genetic construct that carries genetic information. In the context of the present invention, a vector usually refers to a plasmid DNA vector. A vector can refer to any such construct that can be propagated and selected in a host organism. W: Nucleotide code indicating Weak (W=A or T).

Claims

1. A combination system comprising two separate components: a first component is a tag exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon flanked by a 3' intron fragment and a 5' gene of interest (GOI) in an antisense orientation, wherein the first CST is composed of multiple epitopes, and the TEDV does not contain a promoter sequence; a second component is a pool of engineered cells each containing within its genome a tag exchange receiver site (TERS), the TERS encoding a second CST exon distinct from the first CST and encoding a reporter gene in an antisense orientation, the second CST exon being connected by a full-length intron sequence to an exon encoding a transmembrane domain; wherein paired recombinase-mediated cassette exchange (RMCE) elements are contained in the TEDV and TERS, and RMCE between the TEDV and TERS results in exchange of the TEDV-encoded GOI with a reporter element and exchange of the second CST exon with the first CST exon, such that each of the derived engineered cells expresses the first CST and GOI in place of the second CST and reporter gene; The first component is in the 5'→3' direction. a. a first 5' RMCE element encoded within a non-functional non-coding 3' intron fragment; b. a 3' intron fragment, c. an exon containing CST encoded by TEDV in the 5' to 3' direction; d. a first transcription terminator sequence for CST encoded in the 5' to 3' direction; e. A second transcription terminator sequence for the GOI encoded in the 3' to 5' direction as follows: f. a sequence encoding the GOI in the 3' to 5' direction; g. Kozak sequence, h. A second 3' RMCE element TEDV, which includes The CST exon and first transcription terminator are encoded in the antisense direction from the GOI and associated transcription terminator and Kozak sequence, The second component is: a. a transcriptional promoter element; b. Kozak sequence, c. an exon encoding a transmembrane domain; d. 5' intron splice donor site; e. a first 5' RMCE element encoded within the non-functional non-coding 3' intron fragment that is equivalent to and pairs with the 5' RMCE element of TEDV; f. The functional sequence of the 3' intron fragment, including the branch point sequence, polypyrimidine tract, and 3' splice acceptor site. g. An exon containing CST encoded by TERS in the 5'→3' direction; h. a first transcription terminator sequence for CST encoded in the 5' to 3' direction; i. a second transcription terminator sequence for a selected gene encoded in the 3' to 5' direction; j. a sequence encoding a reporter gene in the 3' to 5' direction; K. Kozak sequence, l. a second 3' RMCE element that pairs with the 3' RMCE element of TEDV; m. a second transcription promoter element; TERS, which includes A combination system in which the transmembrane domain exon and the CST exon are encoded in the antisense direction from a reporter gene, a first transcriptional promoter element drives transcription of the combination of the transmembrane domain and CST, and a second transcriptional promoter element drives transcription of the reporter gene.

2. 2. The combined system of claim 1, wherein a first RMCE element of the TEDV is paired with a first RMCE element of the TERS, and a second RMCE element of the TEDV is paired with a second RMCE element of the TERS.

3. The combination system according to claim 1 or 2, wherein the first CST comprises at least two different epitopes for identifying the GOI.

4. The combination system according to any one of claims 1 to 3, wherein the engineered cell contains a single TERS in its genome.

5. The combination system of claim 3 or claim 4 which cites claim 3, wherein the at least two different epitopes are selected from the group comprising HA, MYC and FLAG.

6. Use of the combination system according to any one of claims 1 to 5 for producing a transgenic engineered cell expressing a TEDV-encoded GOI from the TERS locus, said production comprising: a. Producing a TEDV as defined in any one of claims 1 to 5, which encodes a GOI and does not contain a promoter sequence; b) delivering the TEDV to an engineered cell line containing a TERS paired with the TEDV, the TERS being defined in any one of claims 1 to 5, together with a recombinase enzyme that matches the RMCE element encoded by the TERS; c. contacting the cells with two or more affinity reagents specific for TEDV-encoded CST and TERS-encoded CST; d. Selecting the engineered cells based on the attenuation of expression of the reporter gene and CST encoded by TERS and the increase of expression of CST encoded by TEDV to select cells into which the GOI has been integrated. Including, use.

7. The use according to claim 6, wherein the affinity reagent used in step c) is fluorescently labeled to detect the attenuation of expression of CST encoded by TERS and the increase of expression of CST encoded by TEDV, and wherein cells can be separated and selected based on the expression by fluorescence-activated cell sorting.

8. The use according to claim 7, wherein the affinity reagent used in step c) is immobilized on a substrate such that cells expressing TERS-encoded CST or cells expressing TEDV-encoded CST can be depleted or enriched in the target cell population using substrate affinity methods.

9. 10. An in vitro method for producing a plurality of engineered cells from a pool of TEDVs, the engineered cells being derived from the combination system of any one of claims 1 to 5, and expressing a set of GOIs encoded by the TEDVs, comprising: a. Producing a library of two or more TEDVs as defined in any one of claims 1 to 5, each encoding a unique GOI sequence, each having a unique CST encoded by the TEDV, and each not containing a promoter sequence; b) delivering the TEDV library as a pool to an engineered cell line containing a TERS paired with the TEDV, as defined in any one of claims 1 to 5, together with a recombinase enzyme that matches the RMCE element encoded by the TERS; c. contacting the cells with three or more affinity reagents specific for multiple TEDV-encoded CSTs and TERS-encoded CSTs; d. Selecting the engineered cells based on the attenuation of expression of the reporter gene and TERS-encoded CST and the increase of expression of each unique TEDV-encoded CST; An in vitro method comprising:

10. - generating a library of two or more TEDVs as defined in any one of claims 1 to 5, each encoding a unique GOI sequence, each having a unique CST encoded by the TEDV, and each free of a promoter sequence; - delivering the TEDV library as a pool to an engineered cell line containing a TERS as defined in any one of claims 1 to 5 paired with the TEDV, together with a recombinase enzyme that matches the RMCE element encoded by the TERS.

10. An in vitro method for tracing the cell lineage of engineered cells derived from the combination system of any one of claims 1 to 5, wherein the engineered cells express a set of TEDV-encoded GOIs, within a pool of cells produced by the method, comprising: a. contacting cells with two or more affinity reagents specific for multiple CSTs encoded by TEDV; b. Analyzing the content of the engineered cells based on the expression of each unique CST encoded by TEDV; An in vitro method comprising:

11. 6. A tag exchange donor vector (TEDV) as defined in any one of claims 1 to 5, encoding a cell surface tag (CST) exon flanked by 3' intron fragments and a gene of interest (GOI) in antisense orientation, without any promoter sequence, wherein the CST is composed of multiple epitopes, and the TEDV is a. a first 5' RMCE element encoded within a non-functional non-coding 3' intron fragment; b. a 3' intron fragment, c. an exon containing CST encoded by TEDV in the 5' to 3' direction; d. a first transcription terminator sequence for CST encoded in the 5' to 3' direction; e. A second transcription terminator sequence for the GOI encoded in the 3' to 5' direction: f. a sequence encoding the GOI in the 3' to 5' direction; g. Kozak sequence, h. A second 3' RMCE element A tag exchange donor vector (TEDV) comprising the CST exon and first transcription terminator, encoded in the antisense direction from the GOI and associated transcription terminator and Kozak sequence.

12. 10. An engineered cell comprising in its genome a tag exchange receiver site (TERS) as defined in any one of claims 1 to 5, the TERS encodes a cell surface tag (CST) exon and encodes a reporter gene in an antisense orientation, the CST exon being connected to an exon encoding a transmembrane domain by a full-length intron sequence; An engineered cell wherein a recombinase-mediated cassette exchange (RMCE) element is contained in the TERS, and RMCE between the TERS and a tag exchange donor vector (TEDV) defined in any one of claims 1 to 5 and 11 results in exchange of a gene of interest (GOI) encoded by the TEDV with a reporter element.

13. wherein: a. a transcriptional promoter element; b. Kozak sequence, c. an exon encoding a transmembrane domain; d. 5' intron splice donor site; e. a 5' RMCE element encoded within a non-functional, non-coding 3' intron fragment that is equivalent to and pairs with the 5' RMCE element of TEDV; f. The functional sequence of the 3' intron fragment, including the branch point sequence, polypyrimidine tract, and 3' splice acceptor site. g. An exon containing CST encoded by TERS in the 5'→3' direction; h. a first transcription terminator sequence for CST encoded in the 5' to 3' direction; i. a second transcription terminator sequence for a selected gene encoded in the 3' to 5' direction; j. a sequence encoding a reporter gene in the 3' to 5' direction; K. Kozak sequence, k. a second 3' RMCE element; m. A second transcription promoter element Including, The engineered cell of claim 12, wherein the transmembrane domain exon and the CST exon are encoded in an antisense direction from a reporter gene, a first transcriptional promoter element drives transcription of the combination of the transmembrane domain and CST, and a second transcriptional promoter element drives transcription of the reporter gene.

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