Cell surface tag exchange (CSTE) system for tracking and manipulating cells during exchange integration of recombase-mediated nucleic acid

Abstract

The invention relates to a combined system which comprises two independent components, wherein the 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 gene of interest (GOI) in the antisense direction, and the second component is an engineered cell containing a tag-exchange acceptance site (TERS) within its genome, and a tag-exchange acceptance site (TERS) encoding a second CST exon adjoined by a complete intron sequence to an exon encoding a transmembrane domain, and the tag-exchange acceptance site (TERS) also encoding a reporter gene in an antisense direction, in which paired recombinase-mediated cassette exchange (RMCE) elements are incorporated into the TEDV and TERS such that the RMCE elements encode the second CST exon adjacent by a complete intron sequence to the exon encoding the transmembrane domain. The derivative engineered cell is configured such that execution of the RMCE between the TEDV and the TERS results in an exchange of the reporter element into a TEDV-encoded GOI and an exchange of the first CST exon into a second CST exon, such that the derivative engineered cell now expresses the first CST and GOI in place of the second CST and reporter gene.

Description

[0001] This application is a divisional application of PCT application PCT / EP2019 / 068343, filed on July 9, 2019, with the invention name of “Cell Surface Tag Exchange (CSTE) System for Tracking and Manipulating Cells during Recombinase-Mediated Cassette Exchange Integration of Nucleic Acid Sequences into Engineered Recipient Cells”. The date on which the PCT application entered the Chinese national phase is January 8, 2021, and the application number is 201980046307.6. Field of the Invention

[0002] The present invention relates to the field of cell engineering and recombinant DNA technology. In particular, the present invention relates to systems and methods for generating, purifying, tracking and manipulating engineered cells during the integration of a target gene based on recombinase-mediated cassette exchange (RMCE). Background Art

[0003] In the field of genetic manipulation and immunology, recombinase-mediated cassette exchange (RMCE) has become a useful tool for targeted gene modification, see, e.g., Turan et al., Gene (2013): 515 : 1-27. Typically, the post-RMCE method to confirm that the target gene (GOI) is integrated in the correct position requires cell crushing and sample processing. A recently described method overcomes this type of cell crushing requirement and shows that recombination events can be monitored and separated by tracking fluorescent marker protein expression (Phan et al., Sci Rep. (2017): 7 (1): 17771). Although the method of Phan et al. avoids the need for cell crushing, it relies on intracellular protein fluorescence, so the technology is not suitable for high-content screening of integrated constructs because the unique spectral attribute set provided by fluorescent proteins is limited; thus, only a limited number of genes can be simultaneously labeled and detected using the strategy. In addition, intracellular fluorescent proteins cannot be used for physical operations that divide cells by substrate affinity methods (such as magnetic activated cell sorting (MACS)), and substrate affinity methods provide the possibility of high throughput and parallelization during cell engineering workflows or provide the possibility of increasing the efficiency of high-content cell library engineering.

[0004] At present, with the RMCE technology at hand, there is a need for tools for rapid and robust cell engineering in both high-throughput applications and high-content applications, especially in cases where cells have been transfected simultaneously with a pool of vectors containing a pool of individual genes of interest or in cases where it is desired to select cells by substrate affinity methods. There is therefore a clear need to provide efficient and improved methods. SUMMARY OF THE INVENTION

[0006] The present invention relates to a two-component cell surface tag exchange (CSTE) system for tracking and manipulating engineered cells during integration of a gene of interest (GOI) based on recombinase-mediated cassette exchange (RMCE). The first part of the CSTE system represents a tag-exchange donor vector (TEDV) encoding the GOI for integration, together with a first cell surface tag (CST) flanked by a 3' fragment of an intron sequence containing a splice acceptor site. The TEDV construct is flanked by RMCE elements. The second part of the CSTE represents a tag-exchange acceptor site (TERS) contained within the genome of the engineered target cell, encoding a selected gene flanked by a complete intron sequence encoding a RMCE element and a second CST having an in-frame transmembrane domain, the complete intron sequence encoding a RMCE element and a second RMCE element flanking the GOI coding sequence. The TERS-encoded CST is essentially encoded as two exons flanked by a single intron. The paired RMCE elements of TEDV and TERS are so designed that the execution of RMCE between the two constructs results in the exchange of CST and GOI from TEDV into TERS, and the TERS CST and selection genes encoded by TERS are lost accordingly. The CST delivered by TEDV utilizes the transmembrane domain encoded by TERS, wherein the execution of RMCE results in the exchange of unique CST exon sequences. The promoter elements driving CST and selection gene / GOI expression from TERS are foreign to the constructs that have exchanged RMCE, so the sequences delivered by TEDV will usually only be expressed when RMCE is reliably performed. This CSTE system makes it possible to conditionally report the rapid and robust RMCE presented by detecting the affinity epitopes presented by CST on the cell surface, and also makes it possible to physically divide cells by means of affinity methods fixed by substrates. By using multivalent affinity epitopes as CST elements, this CSTE system also makes it possible for multiplexing and lineage tracking of GOI integration processes. The CSTE system represents a powerful tool for rapid and robust cell engineering in high-throughput applications and high-content applications.

[0007] In a first aspect, the present invention provides a combined system comprising two independent components, wherein the first component is a tag-exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon flanked by 3' intronic fragments and a gene of interest (GOI) in antisense orientation, and the second component is an engineered cell, wherein the engineered cell contains a tag-exchange acceptor site (TERS) within its genome, which encodes a second CST exon flanked by a complete intronic sequence to an exon encoding a transmembrane domain, and which also encodes a reporter gene in antisense orientation, wherein paired recombinase-mediated cassette exchange (RMCE) elements are incorporated into the TEDV and the TERS such that execution of the RMCE between the TEDV and the TERS results in exchange of the reporter element for the TEDV-encoded GOI, and exchange of the first CST exon for the second CST exon, such that the derived engineered cell now expresses the first CST and the GOI, replacing the second CST and the reporter gene.

[0008] In one embodiment, the first component is a TEDV comprising

[0009] a. The first RMCE element encoded in the non-coding and 'non-functional' 3' intron fragment - the 5' RMCE element

[0010] b. 3' intron fragment, which contains the branch point sequence, polypyrimidine tract and 3' acceptor splice site

[0011] c. Exon containing the CST encoded by TEDV in the 5' to 3' direction

[0012] d. The first transcription terminator sequence encoding the CST in the 5' to 3' direction

[0013] e. 3' to 5' second transcription terminator of the GOI

[0014] f. Sequence encoding GOI in 3' to 5' direction

[0015] g. Kozak sequence

[0016] h.5' RMCE element

[0017] The CST exon and the first transcriptional terminator are encoded in the antisense direction from the GOI and the associated transcriptional terminator and Kozak sequence.

[0018] In another embodiment, the second component is a TERS comprising

[0019] a. Transcription promoter element

[0020] b. Kozak sequence

[0021] c. Type 2 membrane protein transmembrane domain exon

[0022] d.5' intron splice donor site

[0023] e. A 5' RMCE element encoded in a non-coding and 'non-functional' 3' intronic fragment that is equivalent to and pairs with the 5' RMCE element of TEDV

[0024] f. Functional sequence of the 3' intron fragment, which contains the branch point sequence, polypyrimidine tract and 3' acceptor splice site

[0025] g. An exon containing a CST encoded by TERS in the 5' to 3' direction (which is different from the CST encoded by TEDV)

[0026] h. A transcription terminator sequence encoding a CST in the 5' to 3' direction.

[0027] i. Transcription terminator sequence for 3' to 5' direction

[0028] j. Sequence encoding the selection gene in the 3' to 5' direction

[0029] k. Kozak sequence for selecting efficient translation initiation of gene transcripts

[0030] 1.3'RMCE element

[0031] m. A 3' genomic element responsible for regulating expression of CST transcripts and their tracking, wherein the transmembrane domain exon and the CST exon are encoded in an antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene.

[0032] In a second aspect, the present invention provides a method for generating a derivative engineered cell expressing a GOI encoded by TEDV from a TERS locus, the method comprising:

[0033] a. Generate TEDV encoding GOI

[0034] b. Delivering the TEDV to an engineered cell line containing a paired TERS, along with a recombinase matching the RMCE element encoded therein

[0035] c. contacting the cells with two or more affinity reagents specific for both the CST encoded by TEDV and the CST encoded by TERS

[0036] d. Based on the attenuated expression of the reporter gene and the CST encoded by TERS and the increased expression of the CST encoded by TEDV, the derived engineered cells are selected as a proxy for selecting cells into which the GOI has been integrated.

[0037] In a third aspect, the present invention provides a tag-exchange donor vector (TEDV) encoding a cell surface tag (CST) exon flanked by 3' intronic fragments and a gene of interest (GOI) in antisense orientation.

[0038] In a fourth aspect, the present invention provides an engineered cell comprising a tag-exchange acceptor site (TERS) within its genome, wherein the tag-exchange acceptor site encodes a cell surface tag (CST) exon adjacent to an exon encoding a transmembrane domain by a complete intron sequence and also encodes a reporter gene in an antisense orientation, wherein a recombinase-mediated cassette exchange (RMCE) element is so incorporated into the TERS that execution of RMCE between the TERS and a tag-exchange donor vector (TEDV) results in exchange of the reporter element for a gene of interest (GOI) encoded by the TEDV.

[0039] The present invention provides a system for integrating a GOI into an engineered cell line by means of RMCE, wherein a report of the integration process is conditionally detected by analysis of a co-integrated cell surface tag (CST). In addition, once the GOI is integrated, the unique CST is subsequently lost from the engineered cell line, allowing for dual positive / negative selection of derivative engineered cells expressing the GOI. This 'tag-swap' is highly robust and is the basis for the stringent positive / negative selection conferred by the CSTE system. The second selection gene is also lost from the TERS locus, which enables highly robust dual negative selection and single positive selection during the generation of derivative engineered cells expressing the GOI. Importantly, the 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 they can also be used to physically divide the target cell population by using a substrate-immobilized affinity reagent method. This enables efficient, high-throughput methods for generating derivative engineered cells by dividing cells using methods such as magnetic-activated cell sorting (MACS), compared to more time-consuming and difficult to parallelize methods such as fluorescence-activated cell sorting (FACS). Finally, the CSTE system, replacing intracellular expression of fluorescent proteins and antibiotic resistance genes as markers of integrated processes, provides a much larger selection marker space. In particular, the generation of a large number of unique CST epitopes for conditional selection is readily achievable, in contrast to fluorescent proteins with broad spectral overlap or a highly limited set of antibiotic resistance genes. This broader accessibility of selection markers enables different forms of high-throughput derivative cell generation processes with increased success rates, and further enables high-content methods for generating cell libraries and cell lineage tracking in cell engineering, pathway engineering, and experimental workflows. DETAILED DESCRIPTION OF THE INVENTION

[0041] Figure 1 The overall architecture and operation of the CSTE system are described in . The CSTE system operates as a donor / acceptor pair, where the tag-exchange donor vector (TEDV) serves to deliver the GOI sequence and the cell surface tag (CST) exon to a paired tag-exchange acceptor site (TERS), which is typically contained within the genome of the engineered cell line. The TERS encodes a unique CST exon and a selection gene, which are exchanged with the CST and GOI encoded by the TEDV, respectively.

[0042] The TEDV-encoded CST exon is encoded in the sense (5' to 3') orientation and is in frame with the 3' intronic fragment containing the splice acceptor site, polypyrimidine tract and branch point sequence. Up to 5' of these functional intronic sequences within the 'non-functional' intronic sequences, the first RMCE element is encoded. The GOI is encoded in the antisense (3' to 5') orientation. Up to the 3' end of the construct adjacent to the GOI transcription start site, the second RMCE element is encoded.

[0043] The paired TERS construct carried by the engineered cell line encodes a CST exon that is in frame with the complete intron sequence, in frame with the first RMCE element within the 'non-functional' intron sequence, paired with the first RMCE element of TEDV and in an equivalent context to the first RMCE element of TEDV. Wherein the complete intron sequence encoding this 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 start site and a promoter sequence that drives transcription to the 5' of the TERS construct. Therefore, once translated from the spliced ​​transcript, the TERS-encoded CST is expressed on the cell surface. Similarly, once RMCE is performed between TEDV and TERS, the TEDV-encoded CST exons are exchanged with the TERS-encoded CST exons. This results in the performance of a 'tag-swap', in which the derivative engineered cell line now expresses the TEDV-encoded CST on the cell surface and does not express the TERS-encoded CST. In this process, the TEDV-encoded CST exon acquires the '5' splice donor site, TD domain, transcription start site and promoter sequence from the TERS construct to enable the expression of the TEDV-encoded CST and provide strict site-specific control over the integration of the TEDV-encoded sequence.

[0044] As mentioned above, TERS encodes a selection gene in the antisense (3' to 5') orientation, and a promoter sequence that is capable of driving transcription of this selection gene to the 3' end of the TERS construct. Between the transcription start site of the selection gene and the 3' promoter sequence of TERS, a second RMCE element is encoded that pairs with the second 5' RMCE element encoded in TEDV. Therefore, when RMCE is performed between TEDV and TERS, the GOI encoded by TEDV is exchanged with the selection gene encoded by TERS.

[0045] It is important to note that TEDV does not contain a promoter sequence, and therefore the CST exons and GOIs encoded by TEDV must be complemented with promoter sequences to enable their transcription. Importantly, as outlined above, inclusion of introns for CST exons and TD exon complementation provides even more stringent site selectivity for the integration of sequences encoded by TEDV. In CSTE, the use of intron elements provides the following additional assurance levels: the random integration of constructs will not provide expressible TEDV encoded CSTs on the cell surface. Contrary to the donor vectors containing TD that will deliver the complete CST ORF, the CSTE system only provides CST epitope exons and splice acceptor sites. Therefore, the abnormal random integration of the donor construct will not only require adjacent effective promoter integration, but also will require obtaining II type transmembrane domains or equivalents, with appropriate 5' splice donor sequences. This results in the constructs of random integration being extremely unlikely to express the CSTs encoded by TEDV, thereby minimizing the selection of such random and adverse events during the generation of the derivative engineered cells expressing the delivered GOI.

[0046] The specific architecture of the CSTE system components including TEDV and TERS is described in detail below.

[0047] TERS Architecture

[0048] The TERS contained within the engineered cell line constitutively expresses the TERS-encoded CST and the selection gene, which are exchanged with the TEDV-encoded CST and GOI, respectively, upon RMCE performed with the paired TEDV. Figure 3 The general architecture of TERS is described in .

[0049] TERS generally includes:

[0050] a) Kozak sequence

[0051] b) Exons encoding the transmembrane domain of type 2 membrane proteins.

[0052] c) 5' intron splice donor site.

[0053] d) A 5' RMCE element encoded in a non-coding and 'non-functional' 3' intronic fragment, which is equivalent to and pairs with the 5' RMCE element of TEDV.

[0054] e) A functional sequence of the 3' intron fragment containing the branch point sequence, the polypyrimidine tract and the 3' acceptor splice site.

[0055] f) contains the exons of CST encoded by TERS in the 5' to 3' direction.

[0056] g) A transcription terminator sequence encoding CST in the 5' to 3' direction

[0057] h) Transcription terminator sequence for the 3' to 5' encoded selection gene.

[0058] i) Sequence encoding the selection gene in the 3' to 5' direction

[0059] j) Kozak sequence for selecting efficient translation initiation of gene transcripts

[0060] k) 3' RMCE element

[0061] x) A 5' genomic element responsible for regulating the expression of CST transcripts and their tracking. This genomic element minimally contains the promoter sequence driving CST transcription.

[0062] y) a 3' genomic element responsible for regulating the expression of the selection gene transcript and its tracking. This genomic element contains at least a promoter sequence driving the transcription of the selection gene;

[0063] wherein; providing a) to ensure efficient translation initiation of TERS-encoded CST from adjacent TD / CST transcripts driven by TERS before RMCE and TEDV-encoded CST after RMCE; b) representing the TD that is adjacent to the CST exon by transcript splicing and mediates the insertion of the translated protein construct into the cell cytoplasmic membrane and presents CST to the extracellular space; c) is an RMCE element located within the intron sequence of the TERS construct and equivalent to the 5' RMCE element of TEDV, It mediates CST exon exchange when RMCE is performed within the CSTE system; e) is an intronic splicing acceptor site and related functional sequences, which make it possible to splice introns containing RMCE elements from TERS-driven TD / CST transcripts; f) is an exon that encodes a CST epitope within the TERS site, so that the expressed TD / CST protein presents the epitope to the extracellular space and the epitope can be conditionally detected using a corresponding affinity reagent; g) represents a 5' to 3 ' direction encoding the TD / CST open reading frame; h) represents the transcription terminator of the selection gene open reading frame encoded in the 3' to 5' direction within TERS; i) represents the open reading frame, which encodes the selection gene 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 by TERS before RMCE is performed; k) is an RMCE element located between the transcription start site of the selection gene and the promoter sequence driving the transcription of this open reading frame and is equivalent to the 3' RMCE element of TEDV, which mediates the exchange of the selection gene with the GOI encoded by TEDV when RMCE is performed within the CSTE system; x) minimally represents the promoter sequence that drives the transcription of the TD / CST open reading frame encoded by TERS before RMCE and drives the transcription of the TD / CST encoded by TEDV after RMCE; y) minimally represents the promoter sequence that drives the transcription of the selection gene open reading frame encoded by TERS before RMCE and drives the transcription of the GOI encoded by TEDV after RMCE.

[0064] Figure 3 A schematic diagram showing TERS clonal fragments is shown in FIG.

[0065] TERS basically encodes a fully functional intron sequence containing a 5'RMCE site. This sequence can represent any intron that makes it possible to efficiently splice TD / CST transcripts in the host engineered cell background. The splice acceptor site of TEDV is required to cooperate with the splice donor site of TERS to function. Therefore, the splice acceptor sequences encoded by TEDV and TERS should be the same or equivalent.

[0066] The non-coding and 'non-functional' intronic sequences encoding the RMCE site may also encode other genetic elements such as transcriptional enhancers, transcriptional insulators, unique open reading frames encoding independent components required for the function or reporting of the TERS construct inside the engineered cells, or other unique sequences for transcript and / or construct tracking and quantification.

[0067] In the case of TERS, for example, the encoded TD / CST and selection genes may contain 5' and 3' untranslated regions (UTRs) encoding unique sequences for transcript and / or construct tracking and quantification or conferring regulation of transcript stability.

[0068] The selection gene can be selected from

[0069] a. Antibiotic resistance genes,

[0070] b. Reporter gene

[0071] c. Nutritional deficiency complementation gene,

[0072] d. Inducible suicide gene

[0073] The selection, design and application of such positive selection markers are well known to those skilled in the art. After performing RMCE in the derivative engineered cells expressing the GOI, the parent engineered cells can be eliminated using an inducible suicide gene. The reporter gene can represent a unique TD / CST construct that reports conditionally or constitutively on the cell surface. Multiple selection genes can be incorporated into this part of TERS to enable positive selection during the generation of engineered cell lines and negative selection during the generation of derivative engineered cell lines.

[0074] The 5' and 3' genomic elements minimally encode promoter sequences that mediate transcription of the TD / CST and selection genes. Such promoters may represent components of inducible promoters. These genomic elements may also encode other genetic elements such as transcription enhancers, transcription insulators, distinct open reading frames encoding separate components required for the function or reporting of the TERS construct within the engineered cell, or other unique sequences for construct tracking and quantification.

[0075] It is important to note that in this specification, TD / CST is encoded in the sense (5' to 3') orientation and the selection gene is encoded in the antisense (3' to 5') orientation (e.g. Figure 3 ). This is for clarity of description. There is no practical reason why these orientations could not be flipped in the TERS / TEDV background, provided that the TD / CST construct can incorporate a functional intronic sequence that also encodes a RMCE site that pairs with an equivalent RMCE site within the 3' intronic fragment of TEDV.

[0076] Inclusion of a selection gene is not an absolute requirement for the CSTE system to operate, however it is desirable for triple selection to aid in GOI expression when performing RMCE. Additionally, the generation and maintenance of engineered cell lines using reporters in both sense and antisense orientations ensures that the promoter sequences contained within the 5' and 3' genomic elements are fully functional prior to performing RMCE to generate derivative engineered cells expressing the TEDV-encoded GOI.

[0077] Cell Surface Tag (CST) Architecture

[0078] One of the key functional features of the CSTE system is the use of multiple unique CST constructs to conditionally report the presence of both the exchanged construct and the original TERS construct incorporating the TEDV-encoded sequence once RMCE has been performed.

[0079] In order to achieve a TD-complementary arrangement within the CSTE system, only certain types of TD exons are suitable, namely, those that can be spliced ​​together with the CST exon located 3' thereof while also allowing the CST protein fragment to be exposed extracellularly.

[0080] To simplify presentation of protein products expressing CST epitopes and to minimize the size and complexity of TD exons, TDs from type II membrane proteins are most suitable. Type II TDs are single-pass transmembrane and are located at the N-terminus of the protein, which faces the cytoplasmic side of the plasma membrane, allowing C-terminal CST epitopes to be exposed to the extracellular space. Other TDs, including multi-pass transmembrane TDs, can be used, provided that the spliced ​​CST transcript exposes the CST epitope to the extracellular space.

[0081] The CST exon adjacent to the TD in the spliced ​​transcript has the function of simply presenting a unique epitope extracellularly, wherein the unique epitope can be conditionally detected by using a corresponding (cognate) affinity reagent. Typically, such epitopes include synthetic sequences, sequences encoded by an organism different from the host engineered cell, or sequences from the same host engineered cell but not expressed extracellularly.

[0082] It is often desirable to include a linker domain between the TD and the CST epitope. Since a CST epitope may contain as few as a few amino acids, such a linker region ensures that the CST is available for affinity reagent engagement at the cell surface. Such linker regions may include flexible 'unstructured' regions, or structurally fully folded protein domains.

[0083] The nature of the epitope encoded by the CST exon reflects its uniqueness only 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 generated.

[0084] Generally speaking, the CST construct as a whole should be functionally neutral with respect to cellular functions.Preference is given to transmembrane domains that are simply fused, often via a linker sequence, to an inert epitope structure without external functional domains.

[0085] 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 in order to identify, track, capture a CST epitope or affect its activity. Such affinity reagents will often be labeled with fluorescent, colorimetric, radiometric or other detectable labels to track cells expressing the corresponding (cognate) CST. Alternatively, such affinity reagents can be functionalized onto a substrate to enable a substrate affinity enrichment method to compartmentalize engineered cells expressing CST.

[0086] Engineered cell lines containing TERS

[0087] Cell lines containing TERS are necessarily engineered, given that a synthetic TERS construct has been inserted. Typically, this means that the TERS construct is integrated into the genome of the engineered cell, but may also include other methods for maintaining the TERS construct in the nucleus, such as methods for episomal maintenance of genetic constructs.

[0088] Methods for integrating the construct into the genome of the target mammalian cell are well known to those skilled in the art and can be achieved via homology directed recombination (HDR) and / or random integration, wherein HDR can be promoted by directed mutagenesis at the genomic locus to be subjected to HDR and can be achieved by different means, including but not limited to site-directed mutagenesis by the following means:

[0089] i. Zinc finger nuclease

[0090] ii. CRISPR / Cas9-mediated targeting

[0091] iii. Synthetic transcription activator-like effector nucleases (TALENs)

[0092] Wherein the site-directed nuclease induces site-specific DNA-repair at the target locus via HDR. After such an event, a certain proportion of cells will have incorporated the HDR vector, which can be selected and / or determined by any combination of the following,

[0093] iv. Non-destructive phenotypic expression analysis

[0094] v. Analysis of disruptive phenotypic expression

[0095] vi. Genetic analysis

[0096] Among them, iv and vi are the preferred methods for selecting and confirming successful genomic integration events.

[0097] Alternatively, viral vectors can be used to deliver the desired components in a site-directed or non-targeted manner.

[0098] The method of TERS construct integration is not critical for TERS operation, provided that there are detectable TERS-encoded CSTs at the cell surface and the presence of an expressed selection gene, indicating that the construct is functional for transcription of the contained coding sequences. Indeed, TERS-encoded CSTs and selection genes are convenient selection markers for generating engineered cell lines containing TERS.

[0099] An important aspect to be noted is that for some applications, it is desirable to control the copy number of the integrated TERS construct, particularly those utilizing this ability to generate cell-based arrays of derivative engineered cell lines expressing a single GOI from a library of TEDV constructs and similar 'multiplex' approaches. With this in mind, episomal maintenance of the TERS construct would generally be incompatible with such multiplex approaches to generate derivative engineered cell lines. Methods for elucidating the copy number of a construct within the genome of an engineered cell are well known to those skilled in the art.

[0100] TEDV Architecture

[0101] TEDV encodes CST exons and GOIs that are exchanged with CST exons encoding TERS and a gene of choice, respectively, when RMCE is performed, where TERS is contained within the engineered cell line. Figure 2 The general architecture of TEDV is described in [2].

[0102] TEDV generally includes

[0103] 1) A 5' RMCE element encoded in a non-coding and 'non-functional' 3' intronic fragment.

[0104] 2) The 3' intron fragment contains the branch point sequence, the polypyrimidine tract and the 3' acceptor splice site.

[0105] 3) Contains the exons of CST encoded by TEDV in the 5' to 3' direction.

[0106] 4) A transcription terminator sequence encoding the CST in the 5' to 3' direction.

[0107] 5) Transcription terminator sequence for 3' to 5' encoded GOI.

[0108] 6) A sequence encoding the GOI in the 3' to 5' direction.

[0109] 7) Kozak sequence.

[0110] 8) 3' RMCE element.

[0111] Among them; 1) represents the RMCE element located within the intronic non-coding and non-functional sequence of the 3' intron fragment of the TEDV construct, and is equivalent to the 5' RMCE element of TERS, mediating the CST exon exchange during RMCE within the CSTE system; 2) the 3' intron fragment, which provides a branch point sequence, a polypyrimidine string and a 3' acceptor splicing site for efficient splicing of the TD / CST transcripts that have integrated the TEDV-encoded CST exons after RMCE between TEDV and TERS, so as to generate derivative engineered cells expressing GOI; 3) is an exon encoding a CST epitope within the TEDV construct, so that the expressed TD / CST protein complemented after RMCE between TEDV and TERS presents the epitope to the extracellular space, and the epitope can be conditionally detected by a corresponding (cognate) affinity reagent; 4) represents 5) represents the transcription terminator of the GOI open reading frame encoded in the 5' to 3' direction inside TEDV, wherein once integrated into the TERS site during RMCE, the transcription terminator acts as the transcription terminator of the encoded GOI; 6) represents the open reading frame encoding GOI, which is to be integrated into the engineered cells to generate derivative engineered cells expressing GOI; 7) is provided to ensure efficient translation initiation of GOI transcripts driven from TERS after RMCE; 8) is the RMCE element at the 3' end of the TEDV construct, and is equivalent to the 3' RMCE site of TERS located between the transcription start site of the selected gene and the promoter sequence driving the transcription of this open reading frame.

[0112] Figure 2 A schematic diagram showing TEDV clonal fragments is shown in FIG.

[0113] TEDV can represent a synthetic DNA construct or a cloned construct as produced by methods well known to those skilled in the art. To aid in cloning methods, TEDV can include restriction endonuclease sequences that insert different CST exons and / or GOI sequences into the construct. Those skilled in the art are familiar with the inclusion of such cloning sites.

[0114] In the context of the present invention, TEDV will typically be a plasmid construct that is amplified in bacteria and may therefore also contain an origin of replication and a selection gene.

[0115] TEDV may also represent a construct with sequence motifs that enable packaging of the construct into specific delivery vehicles such as viral vectors well known to those skilled in the art. The use of viral vectors to transduce engineered cells containing TERS will benefit the use of the CSTE system in cells that are otherwise difficult to transfect.

[0116] TEDV may represent an RNA construct that can be reverse transcribed when a suitable reverse transcriptase is provided.

[0117] In the context of the present invention, GOI is defined by any coding or non-coding target nucleic acid sequence. It may include any protein-coding or polypeptide-coding open reading frame, non-protein-coding RNA such as microRNA, short hairpin RNA, tRNA or rRNA.

[0118] Importantly, a GOI can represent a library of variants of a single open reading frame, and TEDVs encoding such GOIs can be compiled into a pooled TEDV library. Due to the reliable multifactorial and conditional reporting that enables precise control of copy number and GOI integration by means of the CSTE system, a high-content library of derived engineered cells can be generated that expresses a single GOI only in each derived engineered cell in the target cell pool. This can be used for a range of cells, pathways, proteins, and specific GOI engineering in the context of living cells (see below).

[0119] RMCE elements and enzymes The use of site-specific recombinases (SSRs) has been demonstrated to be a predictable tool for modifying the cellular genome. There are two major classes of SSRs: Ser integrases and Tyr recombinases, the former including but not limited to ΦC31, γδ-res, ParA, Tn3, Gin, ΦBT1, R4, Bxb1, TP901-1, and the latter including but not limited to Flp, Cre, R. Although both classes are capable of RMCE, Ser integrases have a restrictive effect that relies on initial crossover, so that with the introduction of either, the desired CST and GOI encoded by TEDV or the TEDV vector backbone will appear. Although all Tyr recombinases have not been comprehensively experimentally evaluated, Cre and Flp have been widely used to perform RMCE. In order to integrate a single copy of the TEDV-encoded CST and GOI genetic elements, it is most appropriate to use Flp in conjunction with a well-characterized heterospecific FRT site, because there is a lack of encoded pseudo-FRT sites in the human genome. This is not the case with Cre, which shows promiscuous activity against LoxP genomic pseudo-sites.

[0120] Methods for producing derivative engineered cells

[0121] Those skilled in the art are familiar with the use of RMCE in mammalian cells, where the donor construct can be delivered to the target cell by a range of methods, including but not limited to chemical transfection, electroporation or viral vector delivery. In addition to providing the donor construct, a specific recombinase must also be provided. This recombinase will usually be provided as an independent expression construct that is co-delivered to the target cell along with the donor construct. In some cases, it may be desirable to modify the engineered cells expressing TERS to conditionally express the desired recombinase, increasing the efficiency of the entire process.

[0122] In the case of the CSTE system, once RMCE has been performed by delivering TEDV to engineered cells containing TERS, and followed by a growth phase, the target cell population can be analyzed and or selected for the purpose of generating derivative engineered cells expressing the GOI encoded by the TEDV. This can be implemented in three ways that are inherent to the CSTE system and independent of detecting GOI expression.

[0123] As a reflection of successful tag-exchange, derivative engineered cell lines can be selected based on negative selection of TERS-encoded CSTs and / or positive selection of TEDV-encoded CSTs. These CSTs are conditionally reported and require the addition of a cognate affinity reagent for each CST. Such selection methods can utilize FACS by adding fluorescently labeled affinity reagents and selecting for the desired CST expression profile. Cell division can also be achieved by substrate-based enrichment schemes, such as MACS, due to the extracellular presentation of CST epitopes.

[0124] Further negative selection can be achieved based on a selection gene encoded by TERS, wherein the selection gene represents a reporter gene or an inducible suicide gene. For example, in the case of a fluorescent reporter gene, the reporter gene can utilize FACS, or if the reporter gene itself represents a unique CST, FACS and / or MACS are utilized. Inducible suicide genes can be used to negatively select parental engineered cells from culture; for example, to enrich for derived engineered cells expressing a GOI.

[0125] In summary, dual positive selection and single negative selection provide highly robust selection of derivative engineered cells, which can be conditionally reported and can be used to divide cells with affinity reagents fixed by substrates. If the properties of the GOI encoded by TEDV can be used for non-destructive detection inside derivative engineered cells, this selection can also include positive selection based on the GOI. The above selection can be used in series or in parallel to obtain high-throughput or accurate high-content derivative engineered cell populations.

[0126] These phenotypic analyses will generally be supported by genotypic confirmation of the copy number of the TEDV sequence in the derived engineered cells using methods well known to those skilled in the art. Given the highly standardized CST expression background, where aberrant integration of the TEDV construct leading to cell surface expression of the TEDV-encoded CST is very rare, this genotypic confirmation can also be achieved phenotypically to a certain extent, by excluding cells that highly express the TEDV-encoded CST. Negative selection , especially when using FACS selection methods. In the case of engineered cells expressing multiple TERS sites (see below), copy number control can be similarly achieved based on the extent of expression of the TEDV-encoded CST on the cell surface.

[0127] Method for dividing cells using affinity reagents immobilized on substrates

[0128] The key advantage of the CSTE system is the cell surface exposure of CST epitopes, and therefore the ability to divide cells based on a fixed substrate affinity method. This is particularly useful for high-throughput methods, or for stepwise selection when generating a high-content derivative engineered cell library. Methods well known to those skilled in the art are based on the MACS method of ferromagnetic beads, together with a variety of other affinity reagent functionalized substrates that capture and physically divide cells based on surface epitope specific expression. This type of rapid and conditional positive and / or negative selection based on cell surface CST expression is highly parallel and can therefore be used for high-throughput methods, compared to existing antibiotic resistance selection methods, speed and accuracy are much higher, and compared to FACS selection methods limited to intracellular fluorescent proteins, speed is much faster and cost is reduced. In addition, the ability to positively and negatively select parental engineered cells and derivative engineered cells results in the generation of an accurate high-content library of derivative engineered cells expressing one or more GOIs.

[0129] Methods for cell surface 'barcoding' with multiple and multivalent CSTs

[0130] The advantage of the CSTE system is the increased amount of selectable marker space available. In contrast to standard fluorescent protein reporter and antibiotic resistance gene systems, which are limited to a handful of parallelizable markers, many unique markers can be rapidly generated by using simple epitopes in conjunction with cognate affinity reagents. Not limited solely by the number of available CST epitopes and cognate reagents, multi-epitope CST domains can be easily constructed for all unique combinations of available CST epitopes and cognate detection reagents. The expanded and parallelizable conditional reporter space enables a variety of high-throughput and high-content approaches.

[0131] Multiple TEDV-encoded CST epitopes (each linked to a different GOI) or GOI variant sequences can be used to increase efficiency in high-throughput cell engineering. For example, a pool of such unique vectors can be integrated into a pool of engineered cells expressing TERS. These cells can be manipulated in large quantities by negative selection as outlined above and later divided based on the expression of unique CSTs encoded by TEDVs.

[0132] Similarly, the use of multiple TEDV-encoded CST epitopes enables the generation of medium-content libraries for tracking and selecting living cell lineages. For example, a library of GOIs associated with unique CSTs can be integrated into a pool of derived engineered cells, and the persistence or function of those cell lineages expressing a particular GOI can be tracked across time. This can be applicable to the selection or analysis of proteins, pathways, or cell engineering in culture systems, or can be similarly used in situations where cells are transplanted into living animals and later recovered as living cells for analysis.

[0133] High-content libraries and multi-TERS engineered cells

[0134] Robust positive and negative selection of derived engineered cells expressing your GOI allows for the generation of precise high-content libraries in multiple formats to support protein, pathway, and cell engineering workflows.

[0135] 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, for example with TERS-encoded and TEDV-encoded CST epitopes, will derive a population of derived 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 incorporated variant GOIs.

[0136] Adding multiple TERS with unique CST epitopes and multiple TEDV libraries with unique CST epitopes to a single engineered cell allows for incorporation of multiple variant GOI families into such engineering workflows, e.g., treating enzymes in a biosynthetic pathway as combinatorial variant GOIs. Such scenarios where engineered cells contain multiple TERS can be used to enhance overall expression of a single GOI. Multiple TERS engineered cells will typically require unique heterospecific recombinase sites for each TERS / TEDV pair to ensure efficiency and stability of GOI integration and selection. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] Figure 1 .Composition and operation of the cell surface tag exchange (CSTE) system

[0138] Schematic diagram of the CSTE system. The top panel depicts the system components, with the tag exchange donor vector (TEDV) on the right and engineered cells containing the tag exchange acceptor site (TERS) on the left.

[0139] TEDV encodes RMCE elements (open and filled triangles) at either end of the construct, which pair with the RMCE site contained within TERS. The RMCE element is encoded at the 5' end of the TEDV construct within the sequence representing the 3' intronic fragment (filled triangle), wherein the 3' element of the intron (open circle) containing the branch point sequence, polypyrimidine tract and splice acceptor site is immediately adjacent to the 3' of the RMCE element. Therefore, the RMCE is contained within the 'non-functional' and non-coding intronic sequence, and the 3' intronic fragment contained within the TEDV lacks a 5' splice donor site. Immediately 3' to the splice acceptor site, TEDV encodes the 3' exon of the cell surface tag (CST), which means that the exon encodes the CST portion containing a unique molecule binding motif (grey rectangle). The CST sequence is encoded in the 5' to 3' direction, and this TEDV also encodes the gene of interest (GOI) to be encoded and integrated into TERS in the 3' to 5' direction (hatched rectangle).

[0140] The central portion of TERS contained within the engineered cells encodes elements distinct from those of TEDV, despite having the same architecture. That is, between the RMCE elements, paired with those of TEDV (open and solid triangles), TERS encodes a CST exon distinct from the CST exon encoded by TEDV (chequered rectangle), with a splice acceptor site and associated 3' intronic sequence immediately 5' to this CST (open circles). Similarly, the gene of choice (solid rectangle) is encoded within TERS in an antisense orientation, as is the GOI in TEDV. A promoter sequence (rightward arrow) is included at the 5' end of the construct to drive transcription of the CST. Relative to this promoter sequence 3', a transmembrane domain (TD) exon is encoded (open rectangle), with a 5' intronic sequence immediately 3' (solid circle). This means that the TD exon and CST exon encoded in frame with the intron containing the RMCE element are produced as a continuous transcript, which is spliced ​​to contiguously connect the exons into a single coding mRNA. The TERS-encoded TD-CST product (open / checkered dumbbell) is expressed on the cell surface. At the 3' end of the TERS construct is an independent promoter element that drives transcription of the selection gene (solid rectangle) in a 3' to 5' direction, resulting in expression of the selection gene (solid square).

[0141] Introduction of TEDV into engineered cells containing TERS, together with appropriate expression constructs for recombinases specific for paired RMCE elements in TEDV / TERS, results in the execution of RMCE and the generation of derivative engineered cells expressing the GOI (bottom panel). The TERS-encoded elements have been exchanged with the TEDV-encoded elements. The derivative cell lines thus express the TEDV-encoded CST on the cell surface as a TD-CST product with the original TERS-encoded TD (open / grey dumbbells), and express the GOI (hatched rectangles).

[0142] In summary, performing RMCE between TEDV and TERS results in the generation of derivative engineered cells that have lost expression of the selection gene and the CST encoded by TERS, and have acquired expression of the CST encoded by TEDV and the GOI.

[0143] Figure 2 .Label Switched Delivery Vehicle (TEDV) Architecture

[0144] Schematic diagram of TEDV depicted as a linear construct, with each numbered box representing a key element of the construct architecture. The construct contains the CST encoded by TEDV and a gene of interest (GOI).

[0145] 1) represents a 5' RMCE element encoded in a non-coding and 'non-functional' 3' intronic fragment.

[0146] 2) A functional sequence representing the 3' intron fragment, which contains the branch point sequence, polypyrimidine tract and 3' acceptor splice site.

[0147] 3) represents the exons encoding the CST encoded by TEDV in the 5' to 3' direction.

[0148] 4) represents the transcription terminator sequence for the encoded CST in the 5' to 3' direction.

[0149] 5) represents the transcription terminator sequence for the 3' to 5' encoded GOI.

[0150] 6) represents the sequence encoding GOI in the 3' to 5' direction.

[0151] 7) represents the Kozak sequence for efficient translation initiation of GOI transcripts.

[0152] 8) represents the 3' RMCE element

[0153] Figure 3.Tag-Exchange Receptor Site (TERS) Open Architecture - Schematic diagram of TERS depicted as a linear construct prior to tag exchange, with each numbered letter representing a key element of the construct architecture. The construct contains the TERS-encoded CST and a gene of interest (GOI).

[0154] a) Represents the Kozak sequence for efficient translation initiation of contiguous transmembrane domain (TD) / CST transcripts.

[0155] b) Representation of the exons encoding the type 2 membrane scaffold protein domain.

[0156] c) represents the 5' intron splice donor site.

[0157] d) represents the 5' RMCE element encoded in the non-coding and 'non-functional' 3' intronic fragment, which is equivalent to and pairs with the 5' RMCE element of TEDV.

[0158] e) represents the functional sequence of the 3' intron fragment, which contains the branch point sequence, the polypyrimidine tract and the 3' acceptor splice site.

[0159] f) represents exons encoding the CST encoded by TERS in the 5' to 3' direction.

[0160] g) represents the transcription terminator sequence for the encoded CST in the 5' to 3' direction.

[0161] h) represents the transcription terminator sequence for the 3' to 5' encoded selection gene.

[0162] i) represents the sequence encoding the selected gene in the 3' to 5' direction

[0163] j) represents the Kozak sequence used to select efficient translation initiation of gene transcripts

[0164] k) represents the 3' RMCE element

[0165] x) represents the 5' genomic element responsible for regulating the expression of CST transcripts and their tracking. This genomic element contains at least the promoter sequence driving CST expression.

[0166] y) represents the 5' genomic element responsible for regulating the expression of CST transcripts and their tracking. This genomic element contains at least the promoter sequence driving CST expression.

[0167] Figure 4 Schematic diagram of the exchanged TERS locus of the derived engineered cells after RMCE-mediated exchange of the TERS locus with the element encoded by TEDV. Figure 1As shown in , the elements encoded between the flanking RMCE sites of TEDV are exchanged with the equivalent framework sequences encoded between the flanking RMCE sites of TERS. This generates an exchanged TERS within the derived engineered cell line. The lettered and numbered boxes represent the Figure 2 and Figure 3 The key genetic elements are described in detail in .

[0168] Figure 5 .TEDV byproducts after exchange

[0169] Schematic representation of TEDV byproducts after RMCE-mediated exchange with TERS-encoded elements. Figure 1 As shown in , the elements encoded between the flanking RMCE sites of TEDV are exchanged with the equivalent framework sequences encoded between the flanking RMCE sites of TERS. This generates an exchanged unstable TEDV byproduct during the generation of derivative engineered cell lines. The lettered and numbered boxes represent Figure 2 and Figure 3 The key genetic elements are described in detail in .

[0170] Figure 6 .TERS integration into engineered cells

[0171] a) The TERS cassette encoding the Myc epitope CST and RFP selection genes was integrated into the parental cell line ACL-128 by homologous directed recombination, generating an engineered cell line population ACL-1163. Ten days after electroporation, the cells were stained with anti-Myc antibodies and analyzed by flow cytometry for TERS-encoded selection markers, namely the Myc epitopes CST and RFP. The figure shows single live cells as RFP versus Myc, indicating that among the transfected cells, there is a population of transfectants (Q2 left panel) that show high signals for both RFP and Myc compared to the parental cells (Q2 right panel). b) Cells with high RFP and Myc signals were selected and grown, and representative engineered monoclonal ACL-1163 was analyzed by flow cytometry. The dot plot shows the RFP versus Myc parameters of gated single live cells. Monoclonal ACL-1163 has high RFP and Myc signals as expected (Q2).

[0172] Figure 7.Performing RMCE with TEDV along with tag-exchange and delivering GOI to generate derivative engineered cells Flp recombinase-mediated tag-exchange was performed in ACL-1163 engineered cells. ACL-1163 cells carrying TERS encoding the RFP selection gene and the Myc epitope CST were transfected with TEDV encoding the SBP epitope CST and GOI together 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 showing reduced RFP and Myc signals but high SBP surface expression signals were sorted and expanded as monoclonal clones. a and b) Contour plots showing representative derivative engineered cell monoclonal ACL-3426 (left) compared to parental cells (right). Derivative cells show loss of RFP and Myc signals (top left panel of Q4) compared to parental cells (top right panel of Q4). The derivative engineered cell monoclone ACL-3426 successfully expressed the TEDV-encoded SBP epitope CST, as shown by the increased signal when stained with an anti-SBP antibody (Q5 bottom right panel). These results suggest that the Flp recombinase-mediated tag-swap was successfully performed, as ACL-3426 cells have lost the signals of the RFP marker and the Myc marker as expected and gained SBP surface expression, as expected due to the tag-swap of the CST between TERS and TEDV. c) Immunoblots show three examples of expression of integrated GOIs with C-terminal FLAG-tags. 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. CSTE was performed in three independent experiments, whereby cells were transfected with a construct encoding flp recombinase and with TEDV encoding the SBP epitope CST and RSV-1 P gene (the resulting cell line monoclone was ACL-3374); or with TEDV encoding the SBP epitope CST and RSV-1 N gene (the resulting cell line monoclone was ACL-3386); or with TEDV encoding the SBP epitope CST and RSV-1 M2 long gene (the resulting cell line monoclone was ACL-3433). Proteins were extracted from the monoclones and immunoblotted using mouse anti-Flag antibody. Western blotting results showed 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 each respective cell and the absence of signal in the parental cell line.

[0173] Figure 8 After a successful tag-swapping event, magnetic affinity cell sorting (MACS) is used to enrich tag 2 (SBP) or deplete tag 1 (Myc) from a mixed cell population.

[0174] This figure demonstrates that surface tagging technology can be used to MACS-enrich cells carrying tag 2 or deplete cells carrying tag 1 from a mixed cell population after a successful tag-swap event. A starting mixed population of two engineered monoclonal cell lines, APL-4535 and APL-3015, was used to enrich cells expressing tag 2 (SBP) or deplete tag 1 (Myc) using MACS after a successful tag-swap event. The TERS inside APL-4535 cells encodes the SBP epitope CST and the full-length FLAG tag GOI encoding the intracellular protein; while the TERS inside APL-3015 cells encodes the Myc epitope CST and the RFP selection gene. The two cell populations were mixed together at a percentage of 90% (APL-3015) to 10% (APL-4535) cells and MACS was used to enrich for SBP-positive cells (a) or deplete Myc-positive cells (b) in separate experiments. a) All cells were labeled with anti-SBP-Alexa 488 fluorophore and incubated with anti-mouse IgG iron beads. Samples were subjected to MACS, and fractions were collected before enrichment, flow-through, and binding at three experimental time points. All three fractions were counterstained with anti-c-Myc-Alexa 405, and data were obtained on a BD in-flow instrument. The figure shows the percentage of SBP-positive cells and Myc-positive cells in all three experimental fractions. The fraction before MACS showed that the starting cell population consisted of 90% Myc-positive cells and 10% SBP-positive cells. Successfully enriched SBP-positive cells were shown based on the lack of SBP signal (>0.01% SBP-positive cells) in the flow-through fraction, and after SBP-directed enrichment, 95% of the binding cells were SBP-positive. b) In an independent experiment, all cells were labeled with anti-c-Myc-Alexa405 fluorophores and incubated with anti-mouse IgG iron beads. Using MACS, labeled cells were depleted, and fractions were collected at three experimental time points as shown above. All three fractions were counterstained with anti-SBP-Alexa 488, and data were obtained on a BD in-flow instrument. The figure shows the percentage of SBP-positive cells and Myc-positive cells after Myc-directed depletion in all three experimental fractions. The pre-MACS fraction shows again that the starting cell population consists of 90% Myc-positive cells and 10% SBP-positive cells. According to the decrease in the number of Myc-positive cells in the flow-through fraction (25%) and the increase in the number of SBP-positive cells (75%), the successful depletion of Myc-positive cells is demonstrated. In addition, the combined fraction contains> 90% Myc-positive cells and <5% SBP-positive cells. c) A total of 546 independent monoclonal clones with SBP-positive staining were evaluated to see if they encode the GOI connected to SBP. The figure shows that 96.52% of SBP-positive cells do integrate GOI, while 3.48% of SBP-positive cells do not encode GOI.

[0175] The results show that MACS can be used to enrich for Tag 2 (SBP) positive cells or deplete Tag 1 (Myc) positive cells from a mixed cell population. In addition, the presence of Tag 2 (SBP) can be used as an indicator of successful tag exchange and GOI genomic integration.

[0176] Fig. 9 . Components and operation of the cell surface tag exchange (CSTE) system using barcodes Schematic diagram of the CSTE system using barcodes. a) Depicts the components of the tag exchange donor vector (TEDV). Each TEDV encodes an RMCE element (open and filled triangles) at either end of the construct, which pairs with the RMCE site contained within the TERS. The 5' RMCE element of the TEDV construct is encoded within a sequence representing a 3' intronic fragment (filled triangle), wherein the 3' element of the intron (open circle) containing the branch point sequence, polypyrimidine tract and splice acceptor site is immediately adjacent to the 3' of the RMCE element. Immediately 3' to the splice acceptor site, the TEDV encodes the 3' exon of the cell surface tag (CST), meaning that the exon encodes the CST portion containing a unique molecule binding motif (grey rectangle). The CST sequence is encoded in the 5' to 3' direction, wherein the TEDV also encodes the gene of interest (GOI) to be integrated into the encoded TERS (hatched rectangle) in the 3' to 5' direction.

[0177] Each CST in this example consists of two different epitopes selected from 3 potential unique epitopes (A, B, C), which can be combined into 6 possible unique combinations. AB is effectively equivalent to BA. In this example, the AX combination has been assigned to a GOI family with three variants (GOI ai, a-ii and a-iii), and the BX combination has been assigned to a second GOI family with three variants (GOIb-i, b-ii, b-iii). Collect the various TEDVs in this example. b) Describe the components of the tag exchange acceptor site (TERS). The TERS RMCE element is paired with those of TEDV (hollow triangles and solid triangles). TERS encodes a CST exon different from the CST exon encoded by TEDV (checkered rectangle), with a splice acceptor site and related 3' intron sequence adjacent to the 5' of this CST (hollow circle). Similarly, the selection gene (solid rectangle) is encoded in the antisense direction inside the TERS, and the GOI is also encoded in the TEDV. A promoter sequence (right arrow) is included at the 5' end of the construct to drive CST transcription. 3' to this promoter sequence, a transmembrane domain (TD) exon is encoded (open rectangle) and the 5' intron sequence is immediately 3' to it (filled circle). The TERS-encoded TD-CST product (open / checkered dumbbell) is expressed on the cell surface. At the 3' end of the TERS construct is an independent promoter element that drives transcription of a selection gene (filled rectangle) in a 3' to 5' direction, resulting in expression of the selection gene (filled square). c) Describes the introduction of a TEDV pool into a population of engineered cells containing TERS, along with appropriate expression constructs for recombinases specific for paired RMCE elements in TEDV / TERS, resulting in the execution of RMCE and the generation of derivative engineered cell populations expressing multiple GOIs. During RMCE, TERS-encoded elements are exchanged with TEDV-encoded elements. The derived cell line population thus expresses TEDV-encoded CST on the cell surface as a TD-CST product with the TD encoded by the original TERS (open / grey dumbbells), and expresses the GOI (hatched rectangle). d) Following RMCE, the pool of engineered cells expressing the GOI can be further separated into its individual members by FACS based on the expression of the unique CST barcode. This can be accomplished as a macro-population or individual cell isolates with the desired barcode by single cell sorting methods. Alternatively, the barcodes can be used as a means of identifying the population of interest within the digital data set, and the pool analysis can be performed by FACS without sorting. e) As a proof of principle for barcoding engineered cells using the CSTE system, ACL-5 and ACL-1 cells were transfected with a plasmid encoding CST or a control plasmid without CST using standard methods known to those skilled in the art, using chemical transfection (ACL-5) or electroporation (ACL-1).CST contains 3 unique epitopes: FLAG, MYC and HA (as represented by SEQ ID 20). 48 hours after transfection, cells were harvested and stained with fluorophore-conjugated corresponding (cognate) antibodies: anti-FLAG-PE, anti-MYC-AF647 and anti-HA-AF488. Cells were analyzed by flow cytometry; live cells were gated based on forward scatter (FSC) and side scatter (SSC). The mean fluorescence intensity (MFI) of live cells and the percentage of live cells expressing the corresponding epitope were determined for each of the 3 epitopes. All three epitopes were detected at high ratios and intensities in cells transfected with the plasmid encoding the barcoded CST compared to the empty vector, thus demonstrating the ability of CST to consist of multiple epitopes.

[0178] This application also discloses the following embodiments 1-15.

[0179] 1. A combinatorial system comprising two separate components, wherein the first component is a tag-exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon flanked by 3' intronic fragments and a gene of interest (GOI) in antisense orientation, and the second component is an engineered cell containing within its genome a tag-exchange acceptor site (TERS) encoding a second CST exon different from the first CST, flanked by a complete intronic sequence to an exon encoding a transmembrane domain, and also encoding a reporter gene in antisense orientation, wherein paired recombinase-mediated cassette exchange (RMCE) elements are incorporated into the TEDV and the TERS such that execution of the RMCE between the TEDV and the TERS results in exchange of the reporter element for the GOI encoded by the TEDV, and exchange of the first CST exon for the second CST exon, such that the derived engineered cell now expresses the first CST and the GOI in place of the second CST and the reporter gene.

[0180] 2. According to the combined system according to embodiment 1, wherein the first component is a TEDV, which comprises

[0181] a. First RMCE element

[0182] b.3' intron fragment

[0183] c.CST exon

[0184] d. First transcription terminator

[0185] e. Second transcription terminator

[0186] f.GOI

[0187] g. Kozak sequence

[0188] h. Second RMCE element

[0189] The CST exon and the first transcriptional terminator are encoded in the antisense direction from the GOI and the associated transcriptional terminator and Kozak sequence.

[0190] 3. A combined system according to embodiment 1 or 2, wherein the second component is a TERS comprising

[0191] a. Transcription promoter element

[0192] b. Kozak sequence

[0193] c. Transmembrane domain exon

[0194] d. Intron

[0195] e. The first RMCE element encoded within the intron sequence

[0196] f.CST exon

[0197] g. First transcription terminator

[0198] h. Second transcription terminator

[0199] i. Reporter gene

[0200] j.Kozak sequence

[0201] k. Second RMCE element

[0202] 1. Second transcription promoter element

[0203] Wherein the transmembrane domain exon and the CST exon are encoded in an antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene.

[0204] 4. A combined system according to any one of embodiments 1 to 3, wherein the first RMCE element of the TEDV is paired with the first RMCE element of the TERS, and the second RMCE element of the TEDV is paired with the second RMCE element of the TERS.

[0205] 5. A combinatorial system according to any one of embodiments 1 to 4, wherein each CST exon comprises a sequence encoding one or more molecular affinity tags and the CST encoded by TEDV and TERS are different.

[0206] 6. A combinatorial system according to any one of embodiments 1 to 4, wherein the engineered cell contains a single TERS in its genome.

[0207] 7. A method for generating a derivative engineered cell expressing a GOI encoded by TEDV from a TERS locus, the method comprising:

[0208] a. Generate TEDV encoding GOI

[0209] b. Delivering the TEDV to an engineered cell line containing a paired TERS, along with a recombinase matching the RMCE element encoded therein

[0210] c. Contacting the cells with two or more affinity reagents specific for both TEDV-encoded CST and TERS-encoded CST

[0211] d. Derivative engineered cells were selected based on reduced expression of the reporter gene and TERS-encoded CST and increased expression of TEDV-encoded CST as a surrogate for selecting cells that have integrated the GOI.

[0212] 8. The method according to embodiment 7, wherein the affinity reagent used in step c. is fluorescently labeled, thereby detecting the weakened TERS-encoded CST expression and the increased TEDV-encoded CST expression, thereby making it possible to divide and select cells based on the expression by fluorescence-activated cell sorting.

[0213] 9. A method according to embodiment 7, wherein the affinity reagent used in step c. is fixed to the substrate in such a way that cells expressing CST encoded by TERS can be depleted or cells expressing CST encoded by TEDV can be enriched in the target cell population using a substrate affinity method, such as magnetic-activated cell sorting.

[0214] 10. A method for generating a plurality of derivative engineered cells expressing a series of TEDV-encoded GOIs from a pool of TEDVs, the method comprising,

[0215] a. Generate a library of two or more TEDVs, each encoding a unique GOI sequence and each having a unique TEDV-encoded CST

[0216] b. Delivering the TEDV library as a pool to an engineered cell line containing paired TERS, along with a recombinase matching the RMCE element encoded therein

[0217] c. contacting the cells with three or more affinity reagents specific for both the multiple CSTs encoded by TEDV and the multiple CSTs encoded by TERS

[0218] d. Derivative engineered cells were selected based on reduced expression of the reporter gene and the CST encoded by TERS and increased expression of the unique CST encoded by each TEDV.

[0219] 11. A method for cell lineage tracing of derived engineered cells expressing a series of TEDV-encoded GOIs within a pool of cells as produced by the following steps,

[0220] - Generate two or more TEDV libraries, each encoding a unique GOI sequence, each with a unique TEDV-encoded CST

[0221] - delivering said TEDV library as a pool to an engineered cell line containing paired TERS, together with a recombinase matching the RMCE element encoded therein,

[0222] The method comprises,

[0223] a. Contacting cells with two or more affinity reagents specific for CSTs encoded by multiple TEDVs

[0224] b. Analysis of the content of derived engineered cells based on the expression of CST encoded by each unique TEDV.

[0225] 12. Tag-Exchange Donor Vector (TEDV) encoding a cell surface tag (CST) exon flanked by 3' intronic fragments and a gene of interest (GOI) in antisense orientation.

[0226] 13. The tag-exchange donor vector (TEDV) according to embodiment 12, comprising

[0227] a. First RMCE element

[0228] b.3' intron fragment

[0229] c.CST exon

[0230] d. First transcription terminator

[0231] e. Second transcription terminator

[0232] f.GOI

[0233] g. Kozak sequence

[0234] h. Second RMCE element

[0235] The CST exon and the first transcriptional terminator are encoded in the antisense direction from the GOI and the associated transcriptional terminator and Kozak sequence.

[0236] 14. An engineered cell comprising a tag-exchange acceptor site (TERS) within its genome, encoding a cell surface tag (CST) exon flanked by an intact intronic sequence to an exon encoding a transmembrane domain, and also encoding a reporter gene in an antisense orientation, wherein a recombinase-mediated cassette exchange (RMCE) element is incorporated into the TERS such that execution of RMCE between the TERS and a tag-exchange donor vector (TEDV) results in exchange of the reporter element for a gene of interest (GOI) encoded by the TEDV.

[0237] 15. The engineered cell of embodiment 14, wherein the TERS comprises:

[0238] a. Transcription promoter element

[0239] b. Kozak sequence

[0240] c. Transmembrane domain exon

[0241] d. Intron

[0242] e. First RMCE element

[0243] f.CST exon

[0244] g. First transcription terminator

[0245] h. Second transcription terminator

[0246] i. Reporter gene

[0247] j.Kozak sequence

[0248] k. Second RMCE element

[0249] 1. a second transcription promoter element,

[0250] Wherein the transmembrane domain exon and the CST exon are encoded in antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene.

[0251] The following series of non-limiting embodiments further illustrate the present invention:

[0252] 1. A combinatorial system comprising two separate components, wherein the first component is a tag-exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon flanked by 3' intronic fragments and a gene of interest (GOI) in antisense orientation, and the second component is an engineered cell containing within its genome a tag-exchange acceptor site (TERS) encoding a second CST exon flanked by a complete intronic sequence to an exon encoding a transmembrane domain and also encoding a reporter gene in antisense orientation, wherein paired recombinase-mediated cassette exchange (RMCE) elements are incorporated into the TEDV and the TERS such that execution of the RMCE between the TEDV and the TERS results in exchange of the reporter element for the GOI encoded by the TEDV, and exchange of the first CST exon for the second CST exon, such that the derived engineered cell now expresses the first CST and the GOI, replacing the second CST and the reporter gene.

[0253] 2. A combinatorial system according to embodiment 1, wherein the first cell surface tag (CST) exon is different from the second CST.

[0254] 3. A combined system according to any one of embodiments 1-2, wherein the first component is a TEDV comprising

[0255] a. First RMCE element

[0256] b.3' intron fragment

[0257] c.CST exon

[0258] d. First transcription terminator

[0259] e. Second transcription terminator

[0260] f.GOI

[0261] g. Kozak sequence

[0262] h. Second RMCE element

[0263] The CST exon and the first transcriptional terminator are encoded in the antisense direction from the GOI and the associated transcriptional terminator and Kozak sequence.

[0264] 4. A combined system according to any one of embodiments 1-3, wherein the first component is a TEDV comprising

[0265] a. First RMCE element - 5' RMCE element encoded in a non-coding and 'non-functional' 3' intronic fragment

[0266] b. 3' intron fragment, which contains the branch point sequence, polypyrimidine tract and 3' acceptor splice site

[0267] c. Contains the exon of CST encoded by TEDV in the 5' to 3' direction

[0268] d. The first transcription terminator sequence for the encoded CST in the 5' to 3' direction

[0269] e. Second transcription terminator for 3' to 5' encoded GOI

[0270] f. Sequence encoding GOI in 3' to 5' direction

[0271] g. Kozak sequence

[0272] h.5' RMCE element

[0273] The CST exon and the first transcriptional terminator are encoded in the antisense direction from the GOI and the associated transcriptional terminator and Kozak sequence.

[0274] 5. A combined system according to any one of embodiments 1-3, wherein the first component is a TEDV comprising

[0275] a. First RMCE element

[0276] b.3' intron fragment

[0277] c.CST exon

[0278] d. First transcription terminator

[0279] e. Second transcription terminator

[0280] f.GOI

[0281] g. Kozak sequence

[0282] h. Second RMCE element

[0283] The CST exon and the first transcriptional terminator are encoded in the antisense direction from the GOI and the associated transcriptional terminator and Kozak sequence.

[0284] 6. A combined system according to any one of embodiments 1-5, wherein the second component is a TERS comprising

[0285] a. Transcription promoter element

[0286] b. Kozak sequence

[0287] c. Transmembrane domain exon

[0288] d. Intron

[0289] e. First RMCE element

[0290] f.CST exon

[0291] g. First transcription terminator

[0292] h. Second transcription terminator

[0293] i. Reporter gene

[0294] j.Kozak sequence

[0295] k. Second RMCE element

[0296] 1. Second transcription promoter element

[0297] Wherein the transmembrane domain exon and the CST exon are encoded in an antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene.

[0298] 7. A combined system according to any one of embodiments 1-6, wherein the second component is a TERS comprising

[0299] a. Transcription promoter element

[0300] b. Kozak sequence

[0301] c. Type 2 membrane protein transmembrane domain exon

[0302] d.5' intron splice donor site

[0303] e. a 5' RMCE element encoded in a non-coding and 'non-functional' 3' intronic fragment and which serves as an equivalent to and pairs with the 5' RMCE element of TEDV,

[0304] f. Functional sequence of the 3' intron fragment, which contains the branch point sequence, polypyrimidine tract and 3' acceptor splice site

[0305] g. Contains a CST encoded by TERS in the 5' to 3' direction (which is different from the CST encoded by TEDV

[0306] CST)

[0307] h. A transcription terminator sequence encoding a CST in the 5' to 3' direction.

[0308] i. Transcription terminator sequence for 3' to 5' direction

[0309] j. Sequence encoding the selector gene in the 3' to 5' direction

[0310] k. Kozak sequence for selecting efficient translation initiation of gene transcripts

[0311] 1.3'RMCE element

[0312] m. A 3' genomic element responsible for regulating expression of CST transcripts and their tracking, wherein the transmembrane domain exon and the CST exon are encoded in an antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene.

[0313] 8. A combined system according to any one of embodiments 1-6, wherein the second component is a TERS comprising

[0314] a. Transcription promoter element

[0315] b. Kozak sequence

[0316] c. Transmembrane domain exon

[0317] d. Intron

[0318] e. First RMCE element

[0319] f.CST exon

[0320] g. First transcription terminator

[0321] h. Second transcription terminator

[0322] i. Reporter gene

[0323] j.Kozak sequence

[0324] k. Second RMCE element

[0325] 1. Second transcription promoter element

[0326] Wherein the transmembrane domain exon and the CST exon are encoded in an antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene.

[0327] 9. The combined system according to any one of embodiments 1-8, wherein the first RMCE element of the TEDV is paired with the first RMCE element of the TERS, and the second RMCE element of the TEDV is paired with the second RMCE element of the TERS.

[0328] 10. The combinatorial system according to any one of embodiments 1-9, wherein each CST exon comprises a sequence encoding one or more molecular affinity tags and the CSTs encoded by TEDV and TERS are different.

[0329] 11. A combinatorial system according to any one of embodiments 1-10, wherein the engineered cell contains a single TERS in its genome.

[0330] 12. A method for generating a derivative engineered cell expressing a GOI encoded by TEDV from a TERS locus, the method comprising:

[0331] a. Generate TEDV encoding GOI

[0332] b. Delivering the TEDV to an engineered cell line containing a paired TERS, along with a recombinase matching the RMCE element encoded therein

[0333] c. contacting the cells with two or more affinity reagents specific for both the CST encoded by TEDV and the CST encoded by TERS

[0334] d. Selection of derived engineered cells based on reduced expression of the reporter gene and TERS-encoded CST and increased expression of TEDV-encoded CST as an alternative to selecting cells in which the GOI has been integrated.

[0335] 13. The method according to embodiment 12, wherein the affinity reagent used in step c. is fluorescently labeled, thereby detecting the weakened TERS-encoded CST expression and the increased TEDV-encoded CST expression, thereby making it possible to divide and select cells based on said expression by fluorescence activated cell sorting.

[0336] 14. A method according to embodiment 12, wherein the affinity reagent used in step c. is fixed to the substrate in such a way that cells expressing CST encoded by TERS can be depleted or cells expressing CST encoded by TEDV can be enriched in the target cell population using a substrate affinity method, such as magnetic-activated cell sorting.

[0337] 15. A method for generating a plurality of derivative engineered cells expressing a series of TEDV-encoded GOIs from a pool of TEDVs, the method comprising,

[0338] a. Generate a library of two or more TEDVs, each encoding a unique GOI sequence, and each having a unique TEDV-encoded CST

[0339] b. Delivering the TEDV library as a pool to an engineered cell line containing paired TERS, along with a recombinase matching the RMCE element encoded therein

[0340] c. contacting the cells with three or more affinity reagents specific for both the multiple CSTs encoded by TEDV and the multiple CSTs encoded by TERS

[0341] d. Derivative engineered cells were selected based on reduced expression of the reporter gene and the CST encoded by TERS and increased expression of the unique CST encoded by each TEDV.

[0342] 16. A method for cell lineage tracing of derived engineered cells expressing a series of TEDV-encoded GOIs within a pool of cells as produced by steps a. and b. of embodiment 15, the method comprising:

[0343] a. Contacting cells with two or more affinity reagents specific for CSTs encoded by multiple TEDVs

[0344] b. Analysis of the content of derived engineered cells based on the expression of CST encoded by each unique TEDV.

[0345] 17. A method for cell lineage tracing of derived engineered cells expressing a series of TEDV-encoded GOIs within a pool of cells produced by the following steps,

[0346] - Generate a library of two or more TEDVs, each encoding a unique GOI sequence and each having a unique TEDV-encoded CST

[0347] - delivering said TEDV library as a pool to an engineered cell line containing paired TERS, together with a recombinase matching the RMCE element encoded therein, the method comprising,

[0348] a. Contacting cells with two or more affinity reagents specific for CSTs encoded by multiple TEDVs

[0349] b. Analysis of the content of derived engineered cells based on the expression of CST encoded by each unique TEDV.

[0350] 18. Tag-Exchange Donor Vector (TEDV) encoding a cell surface tag (CST) exon flanked by 3' intronic fragments and a gene of interest (GOI) in antisense orientation.

[0351] 19. The tag-exchange donor vector (TEDV) according to embodiment 18, comprising

[0352] a. First RMCE element

[0353] b.3' intron fragment

[0354] c.CST exon

[0355] d. First transcription terminator

[0356] e. Second transcription terminator

[0357] f.GOI

[0358] g. Kozak sequence

[0359] h. Second RMCE element

[0360] The CST exon and the first transcriptional terminator are encoded in the antisense direction from the GOI and the associated transcriptional terminator and Kozak sequence.

[0361] 20. An engineered cell comprising a tag-exchange acceptor site (TERS) within its genome, encoding a cell surface tag (CST) exon flanked by an intact intronic sequence to an exon encoding a transmembrane domain, and also encoding a reporter gene in an antisense orientation, wherein a recombinase-mediated cassette exchange (RMCE) element is incorporated into the TERS such that execution of RMCE between the TERS and a tag-exchange donor vector (TEDV) results in exchange of the reporter element for a gene of interest (GOI) encoded by the TEDV.

[0362] 21. The engineered cell according to embodiment 20, wherein the TERS comprises:

[0363] a. Transcription promoter element

[0364] b. Kozak sequence

[0365] c. Transmembrane domain exon

[0366] d. Intron

[0367] e. First RMCE element

[0368] f.CST exon

[0369] g. First transcription terminator

[0370] h. Second transcription terminator

[0371] i. Reporter gene

[0372] j.Kozak sequence

[0373] k. Second RMCE element

[0374] 1. a second transcription promoter element,

[0375] Wherein the transmembrane domain exon and the CST exon are encoded in antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene.

[0376] Materials and methods

[0377] Integration of TERS into engineered cell lines

[0378] Electroporation is used to deliver the desired DNA constructs to generate engineered cells that have a single TERS site integrated into the AAVS1 locus by homology-directed recombination.

[0379] Each reaction was performed using the Gene Pulser Xcell TM (Bio-Rad) with the following settings: square wave 285V, pulse duration 12.5ms and 2 pulses with 1s interval, 4x10 cells were electroporated in 500ul of RPMI 1640 (Life Technologies) containing Glutamax-I. 6 For the TERS integration vector (V9.F.5), the DNA concentration used was 15ug / ul, for the plasmid encoding Cas9-P2A-GFP (V1.A.8), it was 10ug / ml, and for the vector encoding the gRNA targeting the integration site AAVS1 site (V2.J.6), it was 7.5ug / ml (Table 3).

[0380] After electroporation, cells were incubated in RPMI 1640 medium containing Glutamax-I + 10% FBS (37°C, 5% CO 2 ) for two days and then analyzed.

[0381] Sorting of polyclonal transfectant cells expressing GFP

[0382] Using FACSJAzz TM Cell sorter (BD biosciences) was used to sort cells electroporated with Cas9-P2A-GFP (V1.A.8) or a plasmid encoding a GFP selection marker (V1.A.4) for transient expression of GFP. Cells were washed and resuspended in a sufficient volume of DPBS before sorting in RPMI 1640 (Life Technologies) containing Glutamax-I with 20% HI-FBS and Anti-Anti100 X.

[0383] Isolation of monoclonal cells stably expressing TERS

[0384] FACS is used to obtain single cells that constitutively express Myc epitope CST and RFP selection genes expressed from the TERS receiving box. To detect Myc CST, cells are stained with anti-Myc antibodies (anti-c-Myc-Alexa 647, SantaCruz) and then sorted. Cells are washed in DPBS and then sorted in RPMI 1640 (Life Technologies) containing Glutamax-I together with 20% HI-FBS and Anti-Anti100 X.

[0385]

[0386] Influx TM GFP and RFP fluorescence were detected on FACS (BD Biosciences) against the filter sets listed in Table 4. Single cells expressing Myc and RFP were sorted into 96-well plates containing 200 ul of growth medium to develop a pool of single clones.

[0387]

[0388]

[0389] Phenotypic screening of monoclonal populations

[0390] Samples of 20,000 cells that had grown into monoclonal populations were transferred to microtiter plates for analysis, and the cells were resuspended in 250 ul DPBS 1X (Life Technologies) and plated on an LRS Fortessa TM (BD biosciences). A 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 100ul staining buffer (DPBS + 2% FBS). The cells were incubated at 4°C for 1 hour and then washed twice with 500ul staining buffer before analysis.

[0391] Genotypic screening of single clones - confirmation of integration in the correct genomic location

[0392] ACL-1163 cells were maintained in normal growth medium RPMI 1640 with Glutamax-I + 10% HI-FBS. Cell confluence was monitored daily until they reached 10-12x10 6 Using QIAamp DNA Minikit (Qiagen), 5x10 6The remaining cells were further expanded and cultured at 3x10 6 Cells were cryopreserved at a density of 10 cells / ml in 70% growth medium + 20% HI-FBS + 10% DMSO.

[0393] To screen and evaluate ACL-1163 monoclonal clones at the molecular level, 20 μl reactions were performed using the components and reaction conditions listed in Tables 5 and 6, respectively. This was accomplished by PCR using a hot start high fidelity DNA polymerase (NEB). To determine if the TERS integration cassette was integrated into the AACS1 locus, primers 15.F.9 and 19.E.7 (Table 7) were used, targeting the left homology arm and the region before the transmembrane domain. The 2.1 kb amplicon indicated that the left homology arm was correctly recombined. First, all components ( Reaction buffer, dNTPs, hot start DNA polymerase, forward and reverse primers, 100 ng DNA template and H 2 O) Prepare PCR master mix. Use C1000Touch TM The PCR reactions were run in a thermal cycler (Bio-Rad). PCR products were run on a 1% agarose gel in 1X TAE buffer using a PowerPacBasic (Bio-Rad), stained with 10,000 dilution of sybersafe and analyzed with Fusion SL (Vilber Lourmat).

[0394]

[0395] Table 6: PCR Cycling Conditions

[0396]

[0397] Table 7. Primers

[0398]

[0399]

[0400] Identification of gene copy number

[0401] DNA of selected monoclones was evaluated for numerous TERS boxes integrated within the cell genome. To achieve this evaluation, droplet digital PCR (ddPCR) was performed using primers and probes specific for the TERS box and reference gene (TRAC) (Table 8). The TERS-specific probe was conjugated with FAM, and the reference gene-specific probe was conjugated with HEX. The ACL-1163 cells were considered to have a diploid integrated copy number for the reference gene (TRAC). Prior to ddPCR, DNA was digested with MfeI (NEB) to separate tandem integrations. The DNA was analyzed using QX200 TM Droplet reader and droplet generator and C1000Touch TM Deep well thermal cycler (Bio-Rad), using ddPCR according to the probe TM The protocol of Supermix (without dUTP) (Bio-Rad) was followed for the reaction setup and cycling conditions. Ch1 for FAM and Ch2 for HEX were used, using QuantaSoft TM The software obtains data.

[0402]

[0403]

[0404] Flp-mediated integration of GOI sequences into derived engineered cell lines

[0405] Electroporation was used to deliver the desired DNA constructs to facilitate Flp recombinase-mediated tag exchange. Each reaction was performed using a Gene Pulser Xcell TM (Bio-Rad) with the following settings: square wave 285V, pulse duration 12.5ms and 2 pulses with 1s interval, 4x10 cells were electroporated in 500ul of RPMI 1640 (Life Technologies) containing Glutamax-I. 6 The DNA concentration used was 7.5 μg / ml for the TEDV vector (V9.F.5), 10 ug / ml for the plasmid encoding FLPO (V12.A.8), and 7.5 μg / ml for the vector encoding GFP to track DNA delivery (V1.A.4) (Table 3).

[0406] After electroporation, cells were incubated in RPMI 1640 medium containing Glutamax-I + 10% FBS (37°C, 5% CO 2 ) for two days, followed by analysis and cell sorting of GFP-positive cells.

[0407] Phenotyping of tag switching

[0408] To determine whether Flp recombinase-mediated tag exchange occurred, cells were stained for surface expression of Myc and SBP and RFP fluorescence intensity was measured.

[0409] 7-10 days after electroporation, cells were harvested and surface stained for SBP and Myc using the following antibodies (anti-SBP-Alexa647 and anti-c-Myc-AlexaPE, SantaCruz). TM GFP and RFP fluorescence were detected on FACS with the filter sets listed in Table 4 (BD Biosciences).

[0410] Single cells expressing SBP but not Myc and RFP were sorted into 96-well plates containing 200 ul of growth medium to develop a panel of single clones.

[0411] 20-24 days after single cell sorting, phenotyping of monoclone was performed. For flow cytometry analysis, cells were transferred from the wells and 300 μl RPMI was added to each tube. Cells were centrifuged at 400 g for 3 minutes at 4 ° C, supernatant was aspirated and cell pellets were resuspended in 25 μl staining mixture or RPMI (unstained control) (staining mixture: anti-SBP-Alexa647 and anti-c-Myc-AlexaPE) 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 ul SB and transferred to 96-well plates to collect data on LSRFortessa. FlowJo was used for analysis.

[0412] Confirmation of GOI expression

[0413] Cells were cultured and after harvesting, cells were lysed with 150 mM NaCl, 50 mM Tris pH 8, 1% CHAPS, 5 mM imidazole, 1 mM PMSF, 1x protease and phosphatase inhibitors (Thermo) at 4 ° C on a shaker for 20 minutes. Lysates were cleaned up by centrifugation at 17000 g for 10 minutes at 4 ° C and subjected to sodium dodecyl sulfate (SDS) gel electrophoresis at 140 V for 1 hour on a 10% acrylamide precast gel (Biorad). The gel was semi-dry transferred to a PVDF membrane (Biorad), which was then blocked in Sea blocking solution 1x (Thermo) in Tris buffered saline / tween 20 (TBST) 1x for 15 minutes and incubated with mouse anti-flag antibody (Sigma) for 2 hours at room temperature. The membrane was washed for 5 minutes in TBST 1x to remove unbound primary antibody, 3 times, and incubated with goat anti-mouse antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. The membrane was finally washed three times in TBST 1x for 5 min to remove unbound secondary antibody, and the HRP signal was realized with ECL substrate (Biorad) and acquired with the Fusion SL Vilber system.

[0414] Confirmation of GOI genomic integration

[0415] Monoclonal cell lines expressing cell surface tag 2 (SBP) were evaluated for co-integration of GOI coding sequences at the molecular level. In a 30 ul reaction, the components and reaction conditions listed in Tables 9 and 10, respectively, were used. This was accomplished by PCR using a hot start high fidelity DNA polymerase (NEB). To determine if the GOI was integrated into the genome, primers 12.G.5 and 21.G.8 targeting the 3′UTR region of the GOI were used (Table 7). First, all components ( Reaction buffer, dNTPs, hot start DNA polymerase, forward and reverse primers, 100 ng DNA template and H 2 O) Prepare PCR master mix. Use C1000Touch TM The PCR reaction was run in a thermal cycler (Bio-Rad). The PCR product was run on a 1% agarose gel in 1X TAE buffer using PowerPac Basic (Bio-Rad), stained with 10,000 dilutions of sybersafe and analyzed with Fusion SL (Vilber Lourmat). A band of the correct size encoding the GOI 3′UTR sequence was confirmed by sanger sequencing.

[0416] Table 9: PCR mixture

[0417]

[0418] Table 10: PCR conditions

[0419]

[0420]

[0421] MACS enrichment / depletion of cells expressing surface tags

[0422] The manufacturer's MACS enrichment / depletion protocol was generally followed (Miltenyi Biotec, #130-047-101, IM0001377.PDF).

[0423] Sample preparation

[0424] The cells were harvested and washed once by centrifugation (300 x g at 4°C for 3 minutes) in staining buffer-M (SB-M - cold Dulbecco's phosphate buffered saline (DPBS), 2% FBS, 2 mM EDTA). The SB-M resuspended cells were filtered through a 40-μm cell strainer to obtain a single cell suspension. The cells were washed with 3 ml of cold SB-M and the cell pellet was collected. 80 μl of staining solution containing appropriate antibodies or dead cell removal reagents were used per 10 million cells, and the samples were incubated at 4°C for 30 minutes. The cells were washed twice with 3 ml of SB-M buffer and pelleted.

[0425] Magnetic bead labeling

[0426] The pelleted cells were labeled with magnetic beads by resuspending the cells in 160 μl SB-M and adding anti-mouse IgG1 MACS beads (Miltenyi) (40 μl MACS beads per 10 million cells). After incubation at 4°C for 20 minutes, the cells were washed with 3 ml SB-M and finally resuspended in 500 ul SB-M.

[0427] Magnetic separation

[0428] LS column (Miltenyi) is placed in the magnetic field of suitable MACS separator (Miltenyi) and rinsed with 3ml SB-M. Also collect the MACS front fraction. Add cell suspension to the column, and collect the flow-through fraction containing unlabeled cells into 15ml conical tube (this is called the flow-through fraction). Remove the column from the separator and place in a suitable collection tube. Add 5ml SB-M to the chromatographic column containing magnetically labeled cells. Use the supplied plunger and apply pressure until the plunger reaches the bottom of the column. Elute the magnetically labeled cells (this is called the combined fraction) from the chromatographic column and the fraction is used in downstream applications.

[0429] Example 1 - TERS integration into engineered cell lines

[0430] This example describes the stable integration of TERS into a cell line to generate an engineered cell line monoclonal ACL-1163 containing a single TERS in the genome.

[0431] In this embodiment, the TERS shown as SEQ ID 1 comprises the following selected genetic elements encoding two genes. The first gene encoded in the sense direction comprises an EF1a promoter upstream of an ORF encoded across 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 is modified to encode a first heterospecific 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 comprises an EF1a promoter upstream of an ORF encoding a fluorescent reporter molecule RFP. The region between the Kozak sequence and the promoter encodes a second heterospecific FRT site (F3). The 3' end of the RFP ORF encodes the bGHpA polyadenylation signal terminator.

[0432] To promote the stable genomic integration of TERS into the genomic safe harbor locus, a plasmid AAVS1 was constructed, in which the DNA element of TERS is flanked by the left and right homologous arms of AAVS1. Each arm contains a sequence of> 500bp homologous to the AAVS1 genomic locus. The stable integration of TERS is achieved by the homology-directed recombination (HDR) process at the AAVS1 genomic safe harbor locus. The ACL-128 cell line was transfected with a plasmid encoding the TERS genetic element flanked by the left and right homologous arms of AAVS1, a plasmid encoding the best gRNA targeting the AAVS1 locus, and a plasmid encoding Cas9-P2A-GFP. After 2 days, FACS sorted cells that were positive for Cas9-P2A-GFP plasmid uptake based on GFP fluorescence. The GFP-sorted cells were further amplified for more than 7 days. The TERS-transfected cells were stained with anti-Myc antibodies and analyzed by flow cytometry for the presence of RFP and Myc epitopes CST ( Figure 6 a) Cells that have integrated TERS into their genome show elevated RFP and Myc signals. These cells are sorted and expanded to represent a single clone. Figure 6A representative monoclonal ACL-1163 is shown in b, which exhibits constant and strong surface expression of RFP and Myc. To determine the integration of the genomic receptor cassette into the targeted AAVS1 site, genomic DNA was extracted from the selected ACL-1163 cell line, and PCR reactions were performed with primers that were internal to the TERS receiver cassette and AAVS1 locus-specific primers (15.F.9 and 19.E.7, see Table 7). PCR amplicons of the expected size were detected (data not shown). In addition, 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 was integrated (data not shown).

[0433] The resulting engineered cell line, ACL-1163, contains a single copy of TERS designed for RMCE with an appropriate paired TEDV.

[0434] Example 2 - RMCE and GOI delivery using TEDV in conjunction with tag-exchange to generate derivative engineered cells This example demonstrates the implementation of the CSTE system as a RMCE-driven reaction between TEDV-encoded sequences and TERS, resulting in CST switching on the cell surface, thus reporting the exchange of constructs incorporating TEDV-encoded sequences and cargo GOI. In this example, the above-described ACL-1163 was used as a target engineered cell line.

[0435] The TEDV in the current embodiment encodes the first heterospecific FRT site (FRT) in the sense direction, a 3' intron fragment containing a branch point sequence, a polypyrimidine tract and a 3' acceptor splice site; exons encoding streptavidin binding peptide (SBP) and SV40 polyadenylation signal terminator. Three independent GOIs derived from respiratory syncytial virus (RSV) are encoded in independent TEDVs in the antisense direction, and each GOIORF is located between the second heterospecific FRT site (F3) and the 3'bGHpA polyadenylation signal terminator. The sequences of SEQ ID 3-SEQ ID 5 represent three independent SBP epitope CSTs and GOIs encoding the TEDV constructs used.

[0436] In this example, the engineered cell line ACL-1163 constructed in Example 1 was electroporated with TEDV (selected from sequences SEQ ID 3-SEQ ID 5) and with a vector encoding the expression of RMCE recombinase (FLPO, V4.1.8, Table 3, SEQ ID 2). The cells were incubated for 7-10 days to allow integration coupling to occur and then stained with anti-Myc antibody and anti-SBP antibody and analyzed by flow cytometry for RFP, Myc and SBP reporter molecule signals. Cells that exhibited reduced RFP and Myc signals, but increased SBP signals (indicating 'tag swapping') were sorted and expanded to represent a batch of single clones. Figure 7 a and Figure 7 Characterization of a representative monoclonal ACL-3426 is depicted in b. To confirm that surface tag exchange was successful, monoclonal ACL-3426 was stained with antibodies against Myc and against SBP and analyzed by flow cytometry for loss of RFP and surface Myc expression ( Figure 7 a) and SBP signal acquisition ( Figure 7 b). In fact, cells subjected to CSTE showed successful tag-exchange by displaying SBP signal and no Myc signal or RFP signal. Parental cells were analyzed in parallel and showed that they had persistently high RFP and Myc signals ( Figure 7 a right panel) and showed a low SBP signal ( Figure 7 bRight panel).

[0437] To demonstrate that GOIORF was integrated and consistently expressed after CSTE, three single clones from independent experiments using the above TEDVs, each encoding a unique RSV-1 GOI, were evaluated by immunoblotting ( Figure 7 c). GOIORF are 3 genes from RSV-1: P, N gene and M2 gene (long), each encoding a Flag tag from TEDV. CSTE was performed, whereby cells were transfected with a construct encoding flp recombinase and with TEDV encoding the SBP epitope CST and RSV-1P gene (the resulting cell line monoclone was ACL-3374); or with TEDV encoding the SBP epitope CST and RSV-1N gene (the resulting cell line monoclone was ACL-3386); or with TEDV encoding the SBP epitope CST and RSV-1M2 long gene (the resulting cell line monoclone was ACL-3433). Proteins were extracted from the monoclones and the samples were immunoblotted using a mouse anti-Flag primary antibody followed by incubation with a goat anti-mouse antibody conjugated to horseradish peroxidase (HRP). The HRP signal was generated using an ECL substrate. A single flag-positive band ( Figure 7c, lanes 2-4), indicating successful CSTE. Parental cells were analyzed in parallel and showed the absence of Flag tag signal ( Figure 7 c Lane 5).

[0438] In summary, this example demonstrates that cell surface tag-swap can be used to conditionally report the presence of the original TERS construct, and the presence of a construct incorporating TEDV-encoded sequences that has been swapped following performance of RMCE, and reports GOI integration and expression, independent of detecting the GOI itself.

[0439] Example 3 - Magnetic Affinity Cell Sorting (MACS) to enrich for Tag 2 (SBP) or deplete Tag 1 (Myc) from a mixed engineered cell population following a successful tag exchange event

[0440] This embodiment shows that surface tag technology can be used for MACS enrichment of tag 2 (SBP) or depletion of tag 1 (Myc). In addition, the presence of tag 2 (SBP) can be used to monitor the incorporation of the target gene (GOI) after the tag exchange event is successful. The initial mixed population of two engineered monoclonal cell lines APL-3015 and APL-4535 was used. These cell populations were mixed with 90% Myc-positive APL-3015 cells and 10% SBP-positive APL-4535 cells. The tag exchange acceptance site (TERS) inside the APL-4535 cells encodes the SBP epitope CST and the full-length GOI with FLAG tag encoding the intracellular protein, while the TERS inside the APL-3015 cells encodes the Myc epitope CST and RFP selection gene. Magnetic affinity cell sorting (MACS) is used to enrich SBP-positive APL-4535 cells from the mixed population. In an independent experiment, MACS is used to deplete Myc-positive APL-3015 cells from the mixed population.

[0441] In the first case, a mixed cell population was labeled with anti-SBP-Alexa 488 fluorophore and subsequently incubated with anti-mouse IgG iron beads. Using MACS, SBP labeled cells were enriched and counterstained with anti-c-Myc-Alexa 405 fluorophore. Figure 8 aDescription of the percentage of SBP-positive cells and Myc-positive cells in three experimental steps: 1) pre-MACS, to confirm the starting ratio of SBP-positive cells and Myc-positive cells in the mixed cell population; 2) flow-through, to assess the percentage of SBP-positive cells and Myc-positive cells not captured by the MACS column when the column was placed in the magnetic field; and 3) bound fraction, to assess the percentage of SBP-positive cells 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 flow instrument. Figure 8 b Demonstrates successful enrichment of SBP-positive cells based on the lack of SBP-positive cells in the flow-through fraction (<0.01), while after SBP-directed enrichment, 95% of bound cells were SBP-positive.

[0442] To demonstrate the use of surface labeling technology to deplete the basic cell line expressing tag 1 (Myc) from the mixed cell population MACS method, all cells were labeled with anti-c-Myc-Alexa 405 fluorophores and incubated with anti-mouse IgG iron beads. Using MACS, the labeled cells were depleted and fractions were collected as described above. All three fractions were counterstained with anti-SBP-Alexa 488 and data were obtained on a BD flow meter. According to the decrease in the number of Myc-positive cells in the flow-through fraction (25%) and the increase in the number of SBP-positive cells (75%), Myc-positive cells were successfully depleted.

[0443] Additionally, to demonstrate the possibility of using the presence of tag 2 (SBP) as a reporter to monitor the incorporation of a GOI following a successful tag swap event, a total of 546 SBP-positive independent clones were evaluated whether they encoded an SBP-linked GOI. Figure 8 The graph in c shows that 96.52% of SBP-positive cells do encode the GOI, whereas 3.48% of SBP-positive cells do not express the GOI.

[0444] The results show that the surface tagging technology is suitable for enriching monoclones expressing Tag 2 (SBP) or depleting the unmodified baseline (monoclones expressing Tag 1 (Myc)) from a mixed cell population after a successful tag swap. In addition, the presence of Tag 2 (SBP) correlates with the incorporation of the GOI after a successful tag swap event and thus the presence of Tag 2 (SBP) on the cell surface can be used as an indicator that the GOI has been successfully delivered into the TERS inside the engineered cell line.

[0445] Example 4 - Composition and Operation of the Cell Surface Tag Exchange (CSTE) System Using Barcodes This example describes a conceptual schematic of barcoding engineered cells expressing a GOI using the CSTE system.

[0446] Fig. 9 Schematic diagram of barcoding cells expressing a GOI using the CSTE system. Panels a and b depict the system components, along with a pool of tag exchange donor vectors (TEDV) and engineered cells containing tag exchange acceptor sites (TERS), respectively.

[0447] Each TEDV encodes RMCE elements at the 5' and 3' ends of the construct, which are paired with the RMCE sites contained within the TERS. The RMCE element at the 5' end of the TEDV construct is encoded within a sequence representing a 3' intron fragment, wherein the 3' end of the RMCE element is immediately adjacent to the 3' element of the intron, comprising a branch point sequence, a polypyrimidine string, and a splice acceptor site. Therefore, the RMCE is contained within the 'non-functional' and non-coding intron sequence, and the 3' intron fragment contained within the TEDV lacks a 5' splice donor site. Immediately adjacent to the 3' of the splice acceptor site, TEDV encodes the 3' exon of a cell surface tag (CST), which means that the exon encodes a CST portion containing a unique molecule binding motif. The CST sequence is encoded in the 5' to 3' direction, and the TEDV also encodes the target gene (GOI) to be encoded and integrated into the TERS in the 3' to 5' direction. Each CST in this example consists of two different epitopes selected from 3 potential unique epitopes (A, B, C), which can be combined into 6 possible unique combinations. Since it is difficult to distinguish the arrangement of each epitope with the existing technology, AB is effectively equivalent to BA. In this example, the AX combination has been assigned to a GOI family with three variants (GOI ai, a-ii and a-iii), and the BX combination has been assigned to a second GOI family with three variants (GOI bi, b-ii, b-iii). Collecting the various TEDVs in this example ( Fig. 9 a).

[0448] The central portion of TERS contained within the engineered cells encodes elements that are distinct from those of TEDV, despite the same architecture. That is, between the RMCE elements, paired with those of TEDV, TERS encodes a CST exon (distinct from the CST encoded by TEDV), with a splice acceptor site and associated 3' intron sequence immediately adjacent to the 5' of this CST. Similarly, the selection gene is encoded within TERS in an antisense orientation, as is the GOI that is also encoded in TEDV. A promoter sequence is included at the 5' end of the construct to drive CST transcription. Relative to the 3' of this promoter sequence, a transmembrane domain (TD) exon is encoded, with a 5' intron sequence immediately adjacent to its 3' end. This means that the TD exon and CST exon encoded in frame with the intron containing the RMCE element are produced as a continuous transcript, which is spliced ​​to contiguously connect the exons into a single encoding mRNA. The TD-CST product encoded by TERS is expressed on the cell surface. At the 3' end of the TERS construct is an independent promoter element that drives transcription of the selection gene in a 3' to 5' direction, resulting in expression of the selection gene ( Fig. 9 b).

[0449] Introduction of a pool of TEDVs into a population of engineered cells containing TERS, along with appropriate expression constructs for recombinases specific for the paired RMCE elements in TEDV / TERS, results in the execution of RMCE and the generation of a derivative engineered cell population expressing the GOI ( Fig. 9 c) The TERS-encoded elements have been exchanged with TEDV-encoded elements. The derived engineered cells thus express the TEDV-encoded CST on the cell surface as a TD-CST product with the original TERS-encoded TD and express the GOI.

[0450] In summary, performing RMCE between a pool of TEDV and TERS results in the generation of a derivative engineered cell pool that has lost expression of the original CST and selection gene encoded by TERS, and each cell in the pool has acquired expression of a CST encoded by TEDV and one of the GOI members from the TEDV pool. The engineered cell pool expressing the GOI can be further analyzed / separated into its individual members by means of FACS, for example, based on the expression of a unique CST barcode ( Fig. 9 d). This can be accomplished, for example, as a macro-population or individual cell isolates with the desired barcodes, using single cell sorting methods. Alternatively, barcodes can be used as a means of identifying populations of interest within a digital data set, and pool analysis can be performed without sorting using FACs, e.g., secondary analysis of cell function, e.g., to correlate variant GOI expression or cell function.

[0451] To demonstrate the concept that CST can be composed of multiple epitopes, ACL-1 and ACL-5 cells were transfected with a plasmid encoding CST or a control plasmid without CST ( Fig. 9 e). CST contains three unique epitopes: FLAG, MYC and HA, and is represented by the sequence SEQ ID 20. Cells were transfected using standard methods known to those skilled in the art, using chemical transfection (ACL-5) or electroporation (ACL-1). After 48 hours, cells were harvested and stained with fluorophore-conjugated corresponding (cognate) antibodies: anti-FLAG-PE, anti-MYC-AF647 and anti-HA-AF488. Cells were analyzed by flow cytometry, and live cells were gated based on forward scatter (FSC) and side scatter (SSC). The mean fluorescence intensity (MFI) of live cells and the percentage of live cells expressing the corresponding epitope were determined for each of the three epitopes. Compared with the empty vector, cells in the sample transfected with the plasmid encoding the barcoded CST detected all three epitopes at a high ratio and intensity, thus showing that CST consists of multiple epitopes.

[0452] The results show that surface tagging technology is suitable for barcoding cell lines because CSTs can be composed of multiple epitopes.

[0453] List of abbreviations

[0454] AAVS1 Adeno-associated virus integration site 1

[0455] APC Antigen presenting cell

[0456] Cas9 CRISPR-associated gene 9

[0457] CMV Cytomegalovirus

[0458] Cre recombinase

[0459] CRISPR Clustered regularly interspaced short palindromic repeats

[0460] CST Cell Surface Labels

[0461] CSTE Cell surface tag exchange

[0462] DMSO Dimethyl sulfoxide

[0463] DNA deoxyribonucleic acid

[0464] DPBS Dulbecco's Phosphate Buffered Saline

[0465] DSB Double-strand break

[0466] dUTP deoxyuridine triphosphate

[0467] EDTA Ethylenediaminetetraacetic acid

[0468] EF1 Elongation factor 1alpha (for eukaryotic translation)

[0469] alpha

[0470] FACS fluorescence activated cell sorting

[0471] FAM Fluorescein amidite

[0472] FBS Fetal Bovine Serum

[0473] FLP flippase

[0474] FRT flippase recognition target

[0475] GFP Green Fluorescent Protein

[0476] GOI Target gene

[0477] gRNA guide RNA

[0478] HLA Human Leukocyte Antigen

[0479] IRES internal ribosome entry site

[0480] MACS Magnetic-Activated Cell Sorting

[0481] NEB New England biolabs

[0482] NHEJ Non-homologous end joining

[0483] ORF Open Reading Frame

[0484] PCR polymerase chain reaction

[0485] RFP Red Fluorescent Protein

[0486] RMCE recombinase-mediated cassette exchange

[0487] RPMI Roswell Park Memorial Institute

[0488] RSV Respiratory syncytial virus

[0489] RT Reverse transcription

[0490] RNA Ribonucleic acid

[0491] SBP Streptavidin Binding Peptide

[0492] SSR site-specific recombinase

[0493] SV40 Simian virus 40

[0494] SV40pA Simian virus 40 poly(A)

[0495] TAA Tumor-associated antigen

[0496] TALEN transcription-like effector nuclease

[0497] TAE Tris-Acetate-EDTA

[0498] T cells T lymphocytes

[0499] TCR T cell receptor

[0500] TCS target coding sequence

[0501] TD transmembrane domain

[0502] TEDV tag exchange donor vector

[0503] TERS tag exchange acceptor site

[0504] rRNA ribosomal RNA

[0505] tRNA transfer RNA

[0506] UTR untranslated region

[0507] ZNF Zinc Finger Nuclease

[0508] List of Definitions Amplicon: A stretch of DNA or RNA that is the source and / or product of artificial amplification using various methods including PCR.

[0509] Antibody: An affinity molecule that is expressed by specialized cells of the immune system (called B cells) and contains two chains. B cells express a very large and very diverse repertoire of antibodies that generally do not bind to self-proteins but can bind to and neutralize pathogens or toxins that would threaten the host. Natural or artificially engineered antibodies are often used as affinity reagents.

[0510] Auxotroph: A mutant organism (especially a bacterium or fungus) that requires specific additional nutrients not required by normal strains.

[0511] Cis-acting elements: non-coding DNA regions that regulate the transcription of nearby ORFs.

[0512] CST: Co-integrated cell surface tags that allow reporting of integration of the gene of interest

[0513] CSTE system: A system that operates as a donor / acceptor pair, where the tag-exchange donor vector serves to deliver the gene sequence of interest and the cell surface tag exon to a paired tag-exchange acceptor site contained within the genome of the engineered cell line. Derivative engineered cells: Engineered cells that have been further genetically modified to exchange the CST and integrate the GOI

[0514] DNA: Deoxyribonucleic acid. The chemical name for the molecules that form the genetic material that codes for genes and proteins.

[0515] Engineered cell: A cell whose genome has been engineered by genetic modification.

[0516] Epitope: A region on an antibody target to which an antibody or other affinity reagent binds.

[0517] Eukaryotic conditional regulatory element: A DNA sequence that can influence the activity of a promoter, which may be induced or repressed under defined conditions.

[0518] Eukaryotic Promoter: A DNA sequence that encodes RNA polymerase binding sites and response elements. The sequence in the promoter region controls the binding of RNA polymerase and transcription factors, so the promoter plays a huge role in determining where and when your gene of interest will be expressed.

[0519] Eukaryotic terminator / signal terminator: A DNA sequence recognized by a protein factor that binds to RNA polymerase II and triggers the termination of the transcription process. It also encodes a polyadenylation signal.

[0520] FACS / Flow Cytometry: Fluorescence Activated Cell Sorting. Flow cytometry is a technique whereby individual cells can be analyzed for the expression of specific cell surface and intracellular markers. A variation of this technique, cell sorting, allows the retrieval of cells carrying a defined set of markers for further analysis.

[0521] Flippase: A recombinant enzyme (flippase, Flp) derived from a 2 μm plasmid of baker's yeast (Saccharomyces cerevisiae).

[0522] Fluorescent (protein) marker: A molecule that has specific extinction and emission characteristics and can be detected using microscopy, FACS and related techniques.

[0523] Genetic cis-regulatory elements: are present on the same DNA molecule as the genes they regulate, whereas trans-regulatory elements can regulate genes that are some distance from the genes from which they are transcribed. Cis-regulatory elements are often binding sites for one or more trans-acting factors.

[0524] Genetic barcoding: DNA barcoding is a taxonomic method that uses short genetic markers in an organism's DNA to identify that organism as belonging to a specific species.

[0525] GOI: Gene of interest defined as any nucleic acid encoding or not encoding a sequence of interest.

[0526] Heterospecific recombinase site: A DNA sequence recognized by a recombinase to promote the crossover of two DNA molecules.

[0527] Homology arm: A stretch of DNA that has nearly identical sequence identity to a complementary homology arm and thus facilitates the exchange of two DNA molecules by a cellular process (homology-directed repair).

[0528] Insulator: A DNA sequence that prevents a gene from being affected by the activation or repression of nearby genes. Insulators also prevent the spread of heterochromatin from silent genes to actively transcribed genes.

[0529] Integration: Physical attachment of a DNA sequence into a chromosome of a cell Internal Ribosome Entry Site (IRES): A DNA sequence that, once transcribed, encodes an RNA element that allows translation to initiate in a cap-independent manner Intron: The noncoding portion of an RNA transcript, or the DNA encoding it, that is spliced ​​out of the RNA molecule before it is translated into protein Branchpoint sequence: The branchpoint nucleotide that initiates a nucleophilic attack 5' to the donor splice site. The free end of the upstream intron then initiates a second nucleophilic attack 3' to the splice acceptor site, releasing the intron as an RNA lariat and covalently combining the two exons

[0530] K is the nucleotide code for ketone (K = G or T)

[0531] Kozak sequence: a short sequence required for efficient translation initiation

[0532] M is the nucleotide code for amino group (M = A or C)

[0533] MACS: Magnetic-activated cell sorting: A cell separation technique in which cells are labeled with affinity molecules containing magnetic particles for separation using a magnetic field.

[0534] Match: At this point, the two components encode genetic elements that direct and restrict the interaction between the complementary components Monoclonal cell line: A defined population of cells that arises from a single ancestral cell through repeated cell replication. N is the nucleotide code for any nucleotide (N = A, T, C, or G)

[0535] Innate: an entity that occurs naturally in a cell Negative selection marker: a selectable marker that confers a negative selection effect on the vector and / or the host organism carrying the vector

[0536] Non-coding gene: a DNA sequence that does not encode proteins and is transcribed into functional non-coding RNA molecules. Origin of replication: a specific sequence in a vector, plasmid or genome that initiates replication.

[0537] ORF: Open reading frame. A stretch of genetic material that encodes the translation framework 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 end or cohesive end.

[0538] PCR: polymerase chain reaction in which specific target DNA molecules are amplified exponentially

[0539] Peptide: A short string of amino acids, usually between 6 and 30 amino acids in length.

[0540] Phenotypic analysis: Analysis of observable characteristics of cells.

[0541] Plasmid: A genetic construct, usually a small circular DNA strand, that can replicate in the cytoplasm of bacteria or protozoa independently of the chromosome.

[0542] Polypeptide: A protein composed of a peptide segment to form a three-dimensional structure.

[0543] Polypyrimidine motif: (C n T n ) motif is rich in pyrimidines and is present upstream of the 3' end of the CAG intron. Positive selection marker: a selectable marker that confers a positive selection effect on a vector and / or a host organism carrying the marker-carrying vector. Primer: a short DNA sequence that allows specific recognition of a target DNA sequence, for example during PCR. Promoter: a regulatory DNA element used for the controlled initiation of gene expression.

[0544] Recombinase: An enzyme that mediates genetic recombination and catalyzes RMCE.

[0545] Reporter element: A genetic element that mediates a reporter signal in the organism or vector carrying the element. It can be used as a positive or negative selection marker.

[0546] Restriction enzyme cleavage sequence: A gene sequence cleaved by a restriction enzyme, which may be foreign or native to the recognition sequence of the restriction enzyme.

[0547] Restriction enzyme recognition sequence: A genetic sequence that a restriction enzyme recognizes and binds to.

[0548] RMCE: Recombinase-mediated cassette exchange. Recombinases catalyze the exchange of genetic material at a genomic acceptor site.

[0549] Splice acceptor site: A DNA sequence at the 3' end of an intron AM, APX CM, or affinity reagent that interacts with cells or TCRsp-based reagents that have a TCRsp on their surface

[0550] Splice donor site: A DNA sequence at the 5' end of an intron Suicide gene: A gene that will mediate cell death in the host organism that carries it. Can be used as a positive or negative selection marker.

[0551] Synthetic: An artificially produced entity.

[0552] TEDV: Tag-exchange donor vector paired with a tag-exchange acceptor site contained within the genome of the engineered cell. Use it to deliver the gene of interest and cell surface tag exons TERS: Paired tag-exchange acceptor sites contained within the genome of the engineered cell line Type II transmembrane domain: A single non-cleavable transmembrane segment of hydrophobic residues close to the N-terminus that acts as a signal / anchor sequence for incorporation, an N-terminal portion attached to the inside of the membrane and a C-terminal portion exposed on the outside of the cell or in the ER lumen.

[0553] Vector: A vector is a genetic construct that carries genetic information. In the context of the present invention, a vector generally describes a plasmid-type DNA vector. A vector may represent any such construct that can be amplified and selected in a host organism.

[0554] W is a nucleotide code indicating weakness (W=A or T).

Claims

1. A combined system comprising two separate components, wherein the first component is a tag-exchange donor vector (TEDV) encoding a first cell surface tag (CST) exon flanked by 3' intronic fragments and a 5' gene of interest (GOI) in antisense orientation, wherein the first CST consists of multiple epitopes, the TEDV does not contain a promoter sequence, and the second component is a pool of engineered cells, each of which contains a tag-exchange acceptor site (TERS) within its genome, which encodes a different sequence from the first CST and is flanked by a complete intronic sequence to a second CST exon encoding an exon of a transmembrane domain and also encoding a reporter gene in an antisense orientation, wherein paired recombinase-mediated cassette exchange (RMCE) elements are incorporated into the TEDV and the TERS such that performance of the RMCE between the TEDV and the TERS results in exchange of the reporter element for a GOI encoded by the TEDV, and exchange of the first CST exon for the second CST exon, such that each derivative engineered cell now expresses the first CST and the GOI in place of the second CST and the reporter gene, wherein the first component is a TEDV that comprises, from 5' to 3' a. The first 5' RMCE element encoded in a non-coding and non-functional 3' intronic fragment b.3' intron fragment c. Exon containing the CST encoded by TEDV in the 5' to 3' direction d. The first transcription terminator sequence encoding the CST in the 5' to 3' direction e. 3' to 5' second transcription terminator sequence of the GOI f. Sequence encoding GOI in 3' to 5' direction g. Kozak sequence h. Second 3' RMCE element wherein the CST exon and the first transcription terminator are encoded in the antisense direction from the GOI and from the associated transcription terminator and Kozak sequence, and The second component is TERS, which contains 5' to 3' a. Transcription promoter element b. Kozak sequence c. Exons encoding protein transmembrane domains d.5' intron splice donor site e. A 5' RMCE element encoded in a non-coding and 'non-functional' 3' intronic fragment that is equivalent to and pairs with the 5' RMCE element of TEDV f. Functional sequence of the 3' intron fragment, which contains the branch point sequence, polypyrimidine tract and 3' acceptor splice site g. Contains the exon of CST encoded by TERS in the 5' to 3' direction h. The first transcription terminator sequence encoding the CST in the 5' to 3' direction. i. 3' to 5' encoding the second transcription terminator sequence of the selection gene j. Sequence encoding the reporter gene in the 3' to 5' direction k.Kozak sequence 1. A second 3' RMCE element that pairs with the 3' RMCE element of TEDV m. Second transcription promoter element Wherein the transmembrane domain exon and the CST exon are encoded in an antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene. 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. A combined system according to claim 1 or 2, wherein the first CST comprises at least two different epitopes for recognizing a GOI.

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

5. The combinatorial system according to any one of claims 1 to 4, wherein at least two epitopes are selected from the group consisting of HA, MYC and FLAG.

6. Use of a combinatorial system as defined in any one of claims 1 to 5 for generating derived engineered cells expressing a GOI encoded by TEDV from a TERS locus, the method comprising, a. Producing a TEDV as defined in any one of claims 1 to 5 and encoding a GOI and not comprising a promoter sequence b. delivering the TEDV to an engineered cell line containing a paired TERS as defined in any one of claims 1 to 5, together with a recombinase matching the RMCE element encoded therein c. Contacting the cells with two or more affinity reagents specific for both TEDV-encoded CST and TERS-encoded CST d. Derivative engineered cells were selected based on reduced expression of the reporter gene and TERS-encoded CST and increased expression of TEDV-encoded CST as a surrogate for selecting cells that have integrated the GOI.

7. The use according to claim 6, wherein the affinity reagent used in step c. is fluorescently labeled, thereby detecting the weakened TERS-encoded CST expression and the increased TEDV-encoded CST expression, thereby making it possible to divide and select cells 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 fixed to the substrate so that cells expressing CST encoded by TERS can be depleted or cells expressing CST encoded by TEDV can be enriched in the target cell population using a substrate affinity method, such as magnetic activated cell sorting.

9. Generating a plurality of derived engineered cells from a combinatorial system as defined in any one of claims 1 to 5 from a pool of TEDVs, the derived engineered cells expressing a series of GOIs encoded by TEDVs, the method comprising, a. 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 TEDV-encoded CST, and each lacking a promoter sequence b. delivering the TEDV library as a pool to an engineered cell line containing paired TERS as defined in any one of claims 1 to 5, together with a recombinase matching the RMCE element encoded therein c. contacting the cells with three or more affinity reagents specific for both the multiple CSTs encoded by TEDV and the multiple CSTs encoded by TERS d. Derivative engineered cells were selected based on reduced expression of the reporter gene and the CST encoded by TERS and increased expression of the unique CST encoded by each TEDV.

10. A method for cell lineage tracing of a derived engineered cell obtained from a combinatorial system as defined in any one of claims 1 to 5, within a pool of cells as produced by the following steps, wherein the derived engineered cell expresses a series of GOIs encoded by TEDVs, - generating two or more TEDV libraries as defined in any one of claims 1 to 5, each encoding a unique GOI sequence, each having a unique TEDV-encoded CST, and each lacking a promoter sequence - delivering said TEDV library as a pool to an engineered cell line containing paired TERS as defined in any one of claims 1 to 5, together with a recombinase matching the RMCE element encoded therein, The method comprises, a. Contacting cells with two or more affinity reagents specific for CSTs encoded by multiple TEDVs b. Analysis of the content of derived engineered cells based on the expression of CST encoded by each unique TEDV.

11. 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' intronic fragments and a gene of interest (GOI) in antisense orientation and containing no promoter sequence, wherein the TEDV comprises from 5' to 3' a. The first 5' RMCE element encoded in a non-coding and non-functional 3' intronic fragment b.3' intron fragment c. Exon containing the CST encoded by TEDV in the 5' to 3' direction d. The first transcription terminator sequence encoding the CST in the 5' to 3' direction e. 3' to 5' second transcription terminator sequence of the GOI f. Sequence encoding GOI in 3' to 5' direction g. Kozak sequence h. Second 3' RMCE element The CST exon and the first transcription terminator are encoded in the antisense direction from the GOI and from the associated transcription terminator and Kozak sequence.

12. An engineered cell comprising within its genome a tag-exchange acceptor site (TERS) as defined in any one of claims 1 to 5, encoding a cell surface tag (CST) exon flanked by a complete intron sequence to an exon encoding a transmembrane domain, and also encoding a reporter gene in an antisense orientation, wherein a recombinase-mediated cassette exchange (RMCE) element is incorporated into the TERS so that execution of RMCE between the TERS and a tag-exchange donor vector (TEDV) as defined in any one of claims 1 to 5 or 11 results in exchange of the reporter element for a gene of interest (GOI) encoded by the TEDV.

13. The engineered cell according to claim 12, wherein the TERS comprises from 5' to 3': a. Transcription promoter element b. Kozak sequence c. Exons encoding protein transmembrane domains d.5' intron splice donor site e. A 5' RMCE element encoded in a non-coding and 'non-functional' 3' intronic fragment that is equivalent to and pairs with the 5' RMCE element of TEDV f. Functional sequence of the 3' intron fragment, which contains the branch point sequence, polypyrimidine tract and 3' acceptor splice site g. Contains the exon of CST encoded by TERS in the 5' to 3' direction h. The first transcription terminator sequence encoding the CST in the 5' to 3' direction. i. 3' to 5' encoding the second transcription terminator sequence of the selection gene j. Sequence encoding the reporter gene in the 3' to 5' direction k.Kozak sequence 1. Second 3' RMCE element m. Second transcription promoter element Wherein the transmembrane domain exon and the CST exon are encoded in an antisense orientation from a reporter gene such that a first transcriptional promoter element drives transcription of the combined transmembrane domain and CST and a second transcriptional promoter element drives transcription of the reporter gene.

Images