Metabolic selection through the asparagine biosynthetic pathway

Abstract

The present disclosure provides isolated mammalian cells that contain reduced or eliminated expression of asparagine synthetase (ASNS). Also provided are methods for preparing such cells and for using such cells for the production of recombinant proteins.

Description

[Technical field]

[0001] The present disclosure relates to mammalian cell lines for use in biological production systems, where the mammalian cell lines have been engineered to have reduced or eliminated expression of components of the asparagine biosynthetic pathway to create an asparagine auxotrophic cell line. [Background technology]

[0002] Development of highly productive clonal cell lines for biomanufacturing typically utilizes one or more well-known selection methods, such as glutamine synthetase (GS for glutamine selection), dihydrofolate receptor (DHFR for hypoxanthine and thymidine selection), antibiotic selection (puromycin, hygromycin, blasticidin, etc.), or P5C synthetase (P5CS-proline selection). While the GS system has become the standard in the industry, there is a need for cell lines that allow for multiple selection methods so that more than one vector can be introduced into the cell line to facilitate the production of molecules such as bispecific antibodies, multispecific antibodies, and other multi-chain enzymes / proteins or proteins / enzymes that require effector proteins for expression. Summary of the Invention

[0003] Various aspects of the present disclosure provide a mammalian cell line for use in biological production systems, wherein the mammalian cell line is engineered to have reduced or eliminated expression of endogenous asparagine synthetase (ASNS) gene. In the absence of endogenously expressed functional ASNS protein, cells require an exogenous source of the amino acid asparagine. Chromosomal ASNS sequences can be inactivated using targeted endonuclease-mediated genomic modification, such as CRISPR ribonucleoprotein (RNP) complexes or zinc finger nucleases. Another aspect of the present disclosure provides a mammalian cell line, wherein the mammalian cell line is engineered to have reduced or eliminated expression of endogenous ASNS gene and reduced or eliminated expression of endogenous glutamine synthetase (GS) gene.

[0004] Another aspect of the present disclosure includes a process for selecting cell lines that have enhanced productivity of the expressed biotherapeutic protein. Another aspect of the present disclosure is to provide a bioproduction system for the expression of bispecific antibodies or biotherapeutic proteins that require the expression of effector proteins more conveniently by utilizing multiple selection systems. The process includes expressing at least one recombinant protein in any of the mammalian cell lines.

[0005] Other aspects and iterations of the disclosure are described in more detail below. [Brief description of the drawings]

[0006] [Figure 1] FIG. 1 shows the role of asparagine synthase in the final step of asparagine biosynthesis.

[0007] [Diagram 2] FIG. 2 depicts the cDNA sequence of ASNS in CHOZN® GS− / − CHO cells, including the preferred ZFN binding site (underlined).

[0008] [Diagram 3]FIG. 3 presents data suggesting that the ASNS gene is present in two copies in the CHOZN® CHO GS− / − cell line genome.

[0009] [Figure 4] FIG. 4 shows the cleavage activity of ZFNs targeted to the binding sites identified in FIG.

[0010] [Diagram 5] FIG. 5 shows the changes in the ASNS coding sequence that occurred as a result of ZFN cleavage.

[0011] [Figure 6] FIG. 6 shows vectors utilized in a glutamine and / or asparagine based selection system.

[0012] [Figure 7] FIG. 7 depicts the endogenous expression control sequences (promoter) of the ASNS gene, including the sequences (underlined) used in the transgenic vector.

[0013] [Figure 8] FIG. 8 shows efficient asparagine selection by ASNS knockout clones.

[0014] [Figure 9] FIG. 9 shows efficient IgG expression driven by an asparagine-based selection system.

[0015] [Figure 10] FIG. 10 represents the growth and viability data of pools expressing GFP, RFP, or GFP+RFP using GS selection, ASNS selection, or GS+ASNS double selection, respectively.

[0016] [Figure 11]FIG. 11 shows efficient selection utilizing CHO cells with both GS and ASNS knocked out and plasmids containing either the GS coding sequence, the ASNS coding sequence, or both the GS coding sequence and the ASNS coding sequence.

[0017] [Figure 12] FIG. 12 represents the fluorescence expression data of pools expressing GFP, RFP, or GFP+RFP using GS selection, ASNS selection, or GS+ASNS double selection, respectively.

[0018] [Figure 13] FIG. 13 represents IgG expression data for pools expressing IgG heavy and light chains using GS selection, ASNS selection, or GS+ASNS double selection, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present disclosure provides mammalian cell lines engineered to have reduced or eliminated expression of the endogenous ASNS gene. Also provided are mammalian cell lines engineered to have reduced or eliminated expression of the endogenous GS gene and reduced or eliminated expression of the endogenous ASNS gene. Methods for producing the engineered cell lines and methods for selecting and using the engineered cell lines to produce recombinant proteins are provided.

[0020] (I) Engineered cell lines Certain aspects of the present disclosure include mammalian cell lines engineered to have reduced or eliminated expression of the endogenous ASNS gene, or alternatively, mammalian cell lines engineered to have reduced or eliminated expression of both the endogenous ASNS gene and the endogenous GS gene.

[0021] The cell lines with reduced or abolished expression of ASNS or reduced expression of ASNS and GS disclosed herein are genetically engineered to modify the chromosomal sequence encoding the ASNS or GS protein. The chromosomal sequence may be modified using targeted endonuclease-mediated genome editing techniques, which are described in detail in section (III) below. For example, the chromosomal sequence may be modified to include at least one nucleotide deletion, at least one nucleotide insertion, at least one nucleotide substitution, or a combination thereof, such that the reading frame is shifted and no protein product is produced (i.e., the chromosomal sequence is inactivated). Inactivating one allele of the chromosomal sequence encoding either ASNS or GS reduces the expression of the protein (i.e., knockdown). Inactivating both alleles of the chromosomal sequence encoding either ASNS or GS eliminates the expression of the protein (i.e., knockout).

[0022] In some embodiments, the expression level of ASNS may be reduced by at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more than about 99%. In other embodiments, the expression level of ASNS may be reduced to a level that is undetectable using techniques standard in the art (e.g., Western immunoblotting assay, ELISA enzyme assay, SDS polyacrylamide gel electrophoresis, etc.).

[0023] Generally, the cell viability, viable cell density, titer, proliferation rate, growth response, cell morphology, levels of apoptosis and autophagy, and / or overall cell health of the engineered cell lines disclosed herein are comparable to that of the unengineered parent cells when supplemented with asparagine and / or an exogenous ASNS coding sequence.

[0024] (a) Cell typeThe engineered cell lines disclosed herein are mammalian cell lines. In some embodiments, the engineered cell lines can be derived from human cell lines. Non-limiting examples of suitable human cell lines include human embryonic kidney cells (HEK293, HEK293T); human connective tissue cells (HT-1080); human cervical cancer cells (HELA); human embryonic retina cells (PER.C6); human kidney cells (HKB-11); human liver cells (Huh-7); human lung cells (W138); human liver cells (Hep G2); human U2-OS osteosarcoma cells, human A549 lung cells, human A-431 epithelial cells, CACO-2 human colorectal adenocarcinoma cells, human pluripotent stem cells, Jurkat human T-lymphocyte cells, or human K562 bone marrow cells. In other embodiments, the engineered cell lines can be derived from non-human cell lines. Suitable cell lines include Chinese hamster ovary (CHO) cells; baby hamster kidney (BHK) cells; mouse myeloma NS0 cells; mouse myeloma Sp2 / 0 cells; mouse mammary C127 cells; mouse embryonic fibroblast 3T3 cells (NIH3T3); mouse B lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse embryonic mesenchymal C3H-10T1 / 2 cells; mouse carcinoma CT26 cells; mouse prostate DuCuP cells. These include mouse breast EMT6 cells, mouse hepatoma Hepa1c1c7 cells, mouse myeloma J5582 cells, mouse epithelial MTD-1A cells, mouse cardiac MyEnd cells, mouse kidney RenCa cells, mouse pancreatic RIN-5F cells, mouse melanoma X64 cells, mouse lymphoma YAC-1 cells, rat glioblastoma 9L cells, rat B lymphoma RBL cells, rat neuroblastoma B35 cells, rat hepatocytes (HTC), buffalo rat liver BRL 3A cells, canine kidney cells (MDCK), canine mammary gland (CMT) cells, rat osteosarcoma D17 cells, rat monocyte / macrophage DH82 cells, monkey kidney SV-40 transformed fibroblast (COS7) cells, monkey kidney CVI-76 cells, or African green monkey kidney (VERO, VERO-76) cells. An extensive list of mammalian cell lines can be found in the American Type Culture Collection catalog (ATCC, Manassas, VA). In some embodiments, the cell lines disclosed herein are other than murine cell lines.In a particular embodiment, the engineered cell line is a CHO cell line. Suitable CHO cell lines are CHO-K1, CHO-K1SV, CHO GS. - / - , CHO S, DG44, DuxB11, and cell lines derived therefrom.

[0025] In various embodiments, the parent cell line may be deficient in glutamine synthetase (GS), dihydrofolate reductase (DHFR), hypoxanthine-guanine phosphoribosyltransferase (HPRT), or a combination thereof. For example, the chromosomal sequences encoding GS, DHFR, and / or HPRT may be inactivated. In certain embodiments, all chromosomal sequences encoding GS, DHFR, and / or HPRT are inactivated in the parent cell line.

[0026] (b) an optional nucleic acid encoding a recombinant protein; In some embodiments, the engineered cell lines disclosed herein may further comprise at least one nucleic acid encoding a recombinant protein. Generally, recombinant proteins are heterologous, meaning that the protein is not native to the cell. The recombinant protein may be a therapeutic protein selected from, but not limited to, an antibody, an antibody fragment, a monoclonal antibody, a humanized antibody, a humanized monoclonal antibody, a chimeric antibody, an IgG molecule, an IgG heavy chain, an IgG light chain, an IgA molecule, an IgD molecule, an IgE molecule, an IgM molecule, a vaccine, a growth factor, a cytokine, an interferon, an interleukin, a hormone, a clotting (or coagulation) factor, a blood component, an enzyme, a therapeutic protein, a nutraceutical protein, a functional fragment or functional variant of any of the above, or a fusion protein comprising any of the above proteins and / or a functional fragment or variant thereof. In certain embodiments, the recombinant protein is a bispecific or multispecific antibody, or a protein that requires an effector protein for expression.

[0027] In some embodiments, the nucleic acid encoding the recombinant protein may be linked to a sequence encoding asparagine synthetase (ASNS), hypoxanthine-guanine phosphoribosyltransferase (HPRT), dihydrofolate reductase (DHFR), and / or glutamine synthetase (GS), so that ASNS, HPRT, DHFR, and / or GS may be used as selection markers. The nucleic acid encoding the recombinant protein may also be linked to a sequence encoding at least one antibiotic resistance gene and / or a sequence encoding a marker protein, such as a fluorescent protein. In some embodiments, the nucleic acid encoding the recombinant protein may be part of an expression construct. The expression construct or vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selection marker sequences, origins of replication, etc. Additional information can be found in "Current Protocols in Molecular Biology" Ausubel et al., John Wiley & Sons, New York, 2003 or "Molecular Cloning: A Laboratory Manual" Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, NY, 3rd edition, 2001.

[0028] In some embodiments, the nucleic acid encoding the recombinant protein can be located extrachromosomally. That is, the nucleic acid encoding the recombinant protein can be transiently expressed from a plasmid, cosmid, artificial chromosome, minichromosome, or another extrachromosomal construct. In other embodiments, the nucleic acid encoding the recombinant protein can be integrated chromosomally into the genome of the cell. The integration can be random or targeted. Thus, the recombinant protein can be stably expressed. In some iterations of this embodiment, the nucleic acid sequence encoding the recombinant protein can be operably linked to a suitable heterologous expression control sequence (i.e., promoter). In other iterations, the nucleic acid sequence encoding the recombinant protein can be located under the control of an endogenous expression control sequence. The nucleic acid sequence encoding the recombinant protein can be integrated into the genome of the cell line using homologous recombination, genome editing via targeted endonucleases, viral vectors, transposons, recombinase-mediated cassette exchange systems, plasmids, and other well-known means. Additional guidance can be found in Ausubel et al. 2003, supra, and Sambrook & Russell, 2001.

[0029] (II) Kit A further aspect of the present disclosure provides a kit for recombinant protein production, wherein the kit comprises any of the engineered cell lines detailed in section (I) above. The kit may further comprise cell growth medium, transfection reagents, plasmid vectors, selection media, recombinant protein purification means, buffers, and the like. The kits provided herein generally comprise instructions for growing the cell line and using it to produce recombinant protein. The instructions included in the kit may be affixed to the packaging material or may be included as a package insert. The instructions are typically, but are not limited to, written or printed materials. Any medium capable of recording such instructions and communicating them to an end user is contemplated by the present disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic disks, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" may include the address of an internet site providing the instructions.

[0030] (III) Methods for preparing engineered cell lines Yet another aspect of the present disclosure provides a method for preparing or engineering a cell line with reduced or abolished expression of ASNS and / or GS, as described in section (I) above. The chromosomal sequence encoding ASNS and / or GS can be knocked down or knocked out using various techniques. In general, engineered cell lines are prepared using a targeted endonuclease-mediated genome modification process. Those skilled in the art will understand that said engineered cell line can also be prepared using a site-specific recombination system, random mutagenesis, or other methods known in the art.

[0031] In general, engineered cell lines are prepared by a method that includes introducing at least one targeting endonuclease or a nucleic acid encoding said targeting endonuclease into a parent cell line of interest, where the targeting endonuclease is targeted to a chromosomal sequence encoding ASNS and / or GS. The targeting endonuclease recognizes and binds to a specific chromosomal sequence, introducing a double-strand break. In some embodiments, the double-strand break is repaired by a non-homologous end joining (NHEJ) repair process. NHEJ is error-prone, and may result in the deletion, insertion, and / or substitution of at least one nucleotide, which disrupts the reading frame of the chromosomal sequence, resulting in no protein product being produced, or in the production of a non-functional protein, for example, via the disruption of the enzyme active site of the protein. In other embodiments, the targeting endonuclease may also be used to alter a chromosomal sequence via a homologous recombination reaction by co-introducing a polynucleotide having substantial sequence identity to a portion of the targeted chromosomal sequence. In such a situation, the double-stranded break introduced by the targeted endonuclease is repaired by a homology-directed repair process in which the chromosomal sequence is exchanged with the polynucleotide in such a way that the chromosomal sequence is altered or altered (e.g., integration of the exogenous sequence).

[0032] (a) Targeting endonuclease A variety of targeting endonucleases can be used to modify the chromosomal sequence encoding ASNS and / or GS.Targeting endonucleases can be naturally occurring proteins or engineered proteins.Suitable targeting endonucleases include, but are not limited to, zinc finger nucleases (ZFNs), CRISPR nucleases, transcription activator-like effector (TALE) nucleases (TALENs), meganucleases, chimeric nucleases, site-specific endonucleases, and artificial targeting DNA double strand break inducers.

[0033] (i) Zinc finger nuclease In certain embodiments, the targeting endonuclease can be a pair of zinc finger nucleases (ZFNs). ZFNs bind to specific target sequences and introduce double-strand breaks at targeted cleavage sites. Typically, ZFNs comprise a DNA binding domain (i.e. zinc finger) and a cleavage domain (i.e. nuclease), each of which is described below.

[0034] DNA-binding domain DNA binding domains or zinc fingers can be engineered to recognize and bind to any nucleic acid sequence. For example, Beerli et al. (2002) Nat. Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001) Nat. Biotechnol. 19:656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:411-416;Zhang et al. (2000) J. Biol. Chem. 275(43):33850-33860;Doyon et al. (2008) Nat. Biotechnol. 26:702-708; and Santiago et al. (2008) Proc. Natl. Acad. Sci. USA 105:5809-5814. Engineered zinc finger binding domains may have novel binding specificities compared to naturally occurring zinc finger proteins. Engineering methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, using a database containing dyadic, triad, and / or tetrad nucleotide sequences and individual zinc finger amino acid sequences, where each dyadic, triad, or tetrad nucleotide sequence is associated with one or more amino acid sequences of zinc fingers that bind to a particular triad or tetrad sequence. See, for example, U.S. Patent Nos. 6,453,242 and 6,534,261, the disclosures of which are incorporated herein by reference in their entirety. As an example, the algorithm described in U.S. Patent No. 6,453,242 can be used to design zinc finger binding domains that target preselected sequences.Alternative methods may be used to design zinc finger binding domains that target specific sequences, such as rational design using nondegenerate recognition code tables (Sera et al. (2002) Biochemistry 41:7074-7081). Publicly available web-based tools for identifying potential target sites in DNA sequences and for designing zinc finger binding domains are known in the art. For example, tools for identifying potential target sites in DNA sequences can be found at zincfingertools.org. Tools for designing zinc finger binding domains can be found at zifit.partners.org / ZiFiT (see also Mandell et al. (2006) Nuc. Acid Res. 34:W516-W523; Sander et al. (2007) Nuc. Acid Res. 35:W599-W605).

[0035] A zinc finger binding domain can be designed to recognize and bind to a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length. In an embodiment, a zinc finger binding domain can be designed to recognize and bind to a DNA sequence ranging from about 9 to about 18 nucleotides in length. Generally, a zinc finger binding domain of a zinc finger nuclease used herein comprises at least three zinc finger recognition regions or zinc fingers, where each zinc finger binds to three nucleotides. In an embodiment, a zinc finger binding domain comprises four zinc finger recognition regions. In another embodiment, a zinc finger binding domain comprises five zinc finger recognition regions. In yet another embodiment, a zinc finger binding domain comprises six zinc finger recognition regions. A zinc finger binding domain can be designed to bind to any suitable target DNA sequence. See, for example, U.S. Patent Nos. 6,607,882; 6,534,261 and 6,453,242, the disclosures of which are incorporated herein by reference in their entirety.

[0036] Exemplary methods for selecting zinc finger recognition regions include phage display and two-hybrid systems, which are described in U.S. Patent Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,410,248, 6,140,466, 6,200,759, and 6,242,568, as well as WO 98 / 37186, WO 98 / 53057, WO 00 / 27878, WO 01 / 88197, and GB 2,338,237, each of which is incorporated herein by reference in its entirety. In addition, enhancement of the binding specificity of zinc finger binding domains is described, for example, in WO 02 / 077227, the entire disclosure of which is incorporated herein by reference.

[0037] Methods for designing and constructing zinc finger binding domains and fusion proteins (and the polynucleotides encoding them) are known to those skilled in the art and are described in detail, for example, in U.S. Patent No. 7,888,121, the entirety of which is incorporated herein by reference. Zinc finger recognition regions and / or multi-finger zinc finger proteins can be linked using suitable linker sequences, including, for example, linkers of 5 amino acids or more in length. For non-limiting examples of linker sequences of 6 amino acids or more in length, see U.S. Patent Nos. 6,479,626, 6,903,185, and 7,153,949, the disclosures of which are incorporated herein by reference in their entirety. The zinc finger binding domains described herein may include suitable linker combinations between the individual zinc fingers of the protein.

[0038] Cleavage Domain Zinc finger nucleases also include a cleavage domain. The cleavage domain portion of zinc finger nuclease can be obtained from any endonuclease or exonuclease. Non-limiting examples of endonucleases from which cleavage domain can be derived include, but are not limited to, restriction endonucleases and homing endonucleases. For example, see New England Biolabs Catalog or Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388. Other enzymes that cleave DNA are known (e.g., S1 nuclease; mung bean nuclease; pancreatic DNase I; micrococcal nuclease; yeast HO endonuclease). See also Linn et al. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993. One or more of these enzymes (or functional fragments thereof) can be used as the source of cleavage domain.

[0039] The cleavage domain can also be derived from an enzyme or portion thereof that requires dimerization for cleavage activity, as described above. Two zinc finger nucleases may be required for cleavage, where each nuclease contains a monomer of an active enzyme dimer. Alternatively, a single zinc finger nuclease may contain both monomers to form an active enzyme dimer. As used herein, an "active enzyme dimer" is an enzyme dimer that can cleave a nucleic acid molecule. The two cleavage monomers may be derived from the same endonuclease (or functional fragments thereof), or each monomer may be derived from a different endonuclease (or functional fragments thereof).

[0040] When two cleavage monomers are used to form an active enzyme dimer, the recognition sites of the two zinc fingers are preferably positioned such that the cleavage monomers are positioned relative to one another in a spatial orientation that allows the cleavage monomers to form an active enzyme dimer (e.g., by dimerization) upon binding of the two zinc fingers to their respective recognition sites. As a result, the near edges of the recognition sites can be about 5 to about 18 nucleotides apart. For example, the near edges can be about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 nucleotides apart. However, it will be understood that any integer number of nucleotides or nucleotide pairs can be interposed between the two recognition sites (e.g., about 2 to about 50 nucleotide pairs or more). For example, the near edges of the recognition sites of zinc finger nucleases, such as those detailed herein, can be 6 nucleotides apart. Typically, the cleavage site is between the recognition sites.

[0041] Restriction endonucleases (restriction enzymes) exist in many species and can bind to DNA (at a recognition site) in a sequence-specific manner and cleave the DNA at or near the binding site. Some restriction enzymes (e.g., Type IIS) cleave DNA at sites distant from the recognition site and have separable binding and cleavage domains. For example, the Type IIS enzyme FokI catalyzes a double-stranded cleavage of DNA 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand. See, e.g., U.S. Patent Nos. 5,356,802, 5,436,150, and 5,487,994; and Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc. Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem. 269:31978-31982. Thus, zinc finger nucleases can include at least one Type IIS restriction enzyme cleavage domain and one or more zinc finger binding domains, which may or may not be engineered. Exemplary Type IIS restriction enzymes are described, for example, in International Publication No. WO 07 / 014,275, the disclosure of which is incorporated herein by reference in its entirety. Additional restriction enzymes also contain separable binding and cleavage domains, and are also contemplated by the present disclosure. See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

[0042] An exemplary Type IIS restriction enzyme in which the cleavage domain is separable from the binding domain is FokI. This particular enzyme is active as a dimer (Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95: 10, 570-10, 575). Thus, in this disclosure, the portion of the FokI enzyme used in the zinc finger nuclease is considered to be the cleavage monomer. Thus, for targeted double-strand cleavage using a Fok cleavage domain, two zinc finger nucleases, each containing a FokI cleavage monomer, may be used to reconstitute an active enzyme dimer. Alternatively, a single polypeptide molecule containing a zinc finger binding domain and two FokI cleavage monomers may be used.

[0043] In certain embodiments, the cleavage domain comprises one or more engineered cleavage monomers that minimize or inhibit homodimerization. As a non-limiting example, amino acid residues at positions 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 of FokI are all targets for affecting dimerization of the Fok cleavage half-domain. An exemplary engineered cleavage monomer of FokI that forms an obligate heterodimer comprises a pair in which a first cleavage monomer comprises a mutation at amino acid residue positions 490 and 538 of FokI, and a second cleavage monomer comprises a mutation at amino acid residue positions 486 and 499.

[0044] Thus, in one embodiment of an engineered truncation monomer, a mutation at amino acid position 490 replaces Glu(E) with Lys(K); a mutation at amino acid residue 538 replaces Iso(I) with Lys(K); a mutation at amino acid residue 486 replaces Gln(Q) with Glu(E); and a mutation at position 499 replaces Iso(I) with Lys(K). Specifically, engineered truncation monomers may be prepared by mutating position 490 from E to K and position 538 from I to K in one truncation monomer to create an engineered truncation monomer designated "E490K:I538K," and mutating position 486 from Q to E and position 499 from I to K in another truncation monomer to create an engineered truncation monomer designated "Q486E:I499K." The engineered truncation monomers are obligate heterodimer mutants in which aberrant cleavage is minimized or eliminated. The engineered truncation monomers can be prepared using suitable methods, for example, by site-directed mutagenesis of the wild-type truncation monomer (FokI), as described in U.S. Patent No. 7,888,121, the entirety of which is incorporated herein.

[0045] Additional Domains In some embodiments, the zinc finger nuclease further comprises at least one nuclear localization sequence (NLS). An NLS is an amino acid sequence that facilitates targeting of zinc finger nuclease proteins into the nucleus to introduce double-strand breaks into target sequences in chromosomes. Nuclear localization signals are known in the art (see, for example, Lange et al., J. Biol. Chem., 2007, 282:5101-5105). Non-limiting examples of nuclear localization signals include PKKKRKV (SEQ ID NO: 1), PKKKRRV (SEQ ID NO: 2), KRPAATKKAGQAKKKK (SEQ ID NO: 3), YGRKKRRQRRR (SEQ ID NO: 4), RKKRRQRRR (SEQ ID NO: 5), PAAKRVKLD (SEQ ID NO: 6), RQRRNELKRSP (SEQ ID NO: 7), VSRKRPRP (SEQ ID NO: 8), PPKKARED (SEQ ID NO: 9), PQPKKKPL (SEQ ID NO: 10), SALIKKKKKKMAP (SEQ ID NO: 11), PKQKKRK (SEQ ID NO: 12), RKLKKKIKKL (SEQ ID NO: 13), REKKKFLKRR (SEQ ID NO: 14), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15), RKCLQAGMNLEARKTKK (SEQ ID NO: 16), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 17), and RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 18). The NLS can be located at the N-terminus, C-terminus, or internally of the zinc finger nuclease.

[0046] In further embodiments, the zinc finger nuclease may also include at least one cell membrane permeation domain. Examples of suitable cell membrane permeation domains include, but are not limited to, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 19), PLSSIFSRIGDPPKKKRKV (SEQ ID NO: 20), GALFLGWLGAAGSTMGAPKKKRKV (SEQ ID NO: 21), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 22), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 23), YARAAARQARA (SEQ ID NO: 24), THLPRRRRR (SEQ ID NO: 25), GGRRRRR (SEQ ID NO: 26), RRQRRRTSKLMKR (SEQ ID NO: 27), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 28), KALAWEAKLAKALAKHLAKALAKALKCEA (SEQ ID NO: 29), and RQIKIWFQNRRMKWKK (SEQ ID NO: 30). The cell membrane permeation domain can be located at the N-terminus, C-terminus, or internally of the zinc finger nuclease.

[0047] In yet other embodiments, the zinc finger nuclease may further comprise at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags. In certain embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami). Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), or any other suitable fluorescent protein. In another embodiment, the marker domain may be a purification tag and / or an epitope tag.Suitable tags include, but are not limited to, poly(His) tag, FLAG (or DDK) tag, Halo tag, AcV5 tag, AU1 tag, AU5 tag, biotin carboxyl carrier protein (BCCP), calmodulin binding protein (CBP), chitin binding domain (CBD), E tag, E2 tag, ECS tag, eXact tag, Glu-Glu tag, glutathione-S-transferase (GST), HA tag, HSV tag, KT3 tag, maltose binding protein (MBP), MAP tag, Myc tag, NE tag, NusA tag, PDZ tag, S tag, S1 tag, SBP tag, Softag 1 tag, Softag 3 tag, Spot tag, Strep tag, SUMO tag, T7 tag, tandem affinity purification (TAP) tag, thioredoxin (TRX), V5 tag, VSV-G tag, and Xa tag. The marker domain can be located at the N-terminus, C-terminus, or internally of the zinc finger nuclease.

[0048] At least one nuclear localization signal, at least one cell membrane permeation domain, and / or at least one marker domain may be directly linked to the zinc finger nuclease via one or more chemical bonds (e.g., covalent bonds). Alternatively, at least one nuclear localization signal, at least one cell membrane permeation domain, and / or at least one marker domain may be indirectly linked to the zinc finger nuclease via one or more linkers. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, p-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymer linkers (e.g., PEG). Linkers may include one or more spacer groups, including, but not limited to, alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, etc. The linker may be neutral, or may have a positive or negative charge. In addition, the linker may be cleavable, such that the covalent bond of the linker that connects the linker to another chemical group can be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, catalyst, or enzyme. In some embodiments, the linker may be a peptide linker. The peptide linker may be a flexible amino acid linker or a rigid amino acid linker. Further examples of suitable linkers are well known in the art, and programs for designing linkers are also readily available (Crasto et al., Protein Eng., 2000, 13(5):309-312).

[0049] (ii) CRISPR ribonucleoproteins (RNPs) In other embodiments, targeting endonuclease can be clustered regularly interspaced short palindromic repeats (CRISPR) nuclease.CRISPR nuclease is the RNA-guided nuclease derived from bacterial or archaeal CRISPR / CRIPSR-associated (Cas) system.CRISPR RNP system comprises CRISPR nuclease and guide RNA.

[0050] Nucleases CRISPR nucleases can be derived from Type I (i.e., IA, IB, IC, ID, IE, or IF), Type II (i.e., IIA, IIB, or IIC), Type III (i.e., IIIA or IIIB), Type V, or Type VI CRISPR systems present in a variety of bacteria and archaea. For example, CRISPR nucleases have been shown to be effective in the detection and characterization of Streptococcus sp. (e.g., S. pyogenes, S. thermophilus, S. pasteurianus), Campylobacter sp. (e.g., Campylobacter jejuni), Francisella sp. (e.g., Francisella novicida), Acaryochloris sp., Acetohalobium sp., Acidaminococcus sp., Acidithiobacillus sp., and the like. sp., Alicyclobacillus sp., Allochromatium sp., Ammonifex sp., Anabaena sp., Arthrospira sp., Bacillus sp., Burkholderiales sp., Caldicelulosiruptor sp., Candidatus sp., Clostridium sp., Crocosphaera sp., Cyanothece sp., Exiguobacterium sp., Finegoldia sp. sp., Ktedonobacter sp., Lachnospiraceae sp.), Lactobacillus sp., Lyngbya sp., Marinobacter sp., Methanohalobium sp., Microscilla sp., Microcoleus sp., Microcystis sp., Natranaerobius sp., Neisseria sp., Nitrosococcus sp., Nocardiopsis sp., Nodularia sp., Nostoc sp., Oscillatoria sp., Polaromonas sp. sp.), Pelotomaculum sp., Pseudoalteromonas sp., Petrotoga sp., Prevotella sp., Staphylococcus sp., Streptomyces sp., Streptosporangium sp., Synechococcus sp., Thermosipho sp., or Verrucomicrobia sp. In other embodiments, the CRISPR nuclease may be derived from an archaeal CRISPR system, a CRISPR / CasX system, or a CRISPR / CasY system (Burstein et al., Nature, 2017, 542(7640):237-241).

[0051] In some embodiments, the CRISPR nuclease can be derived from a type II CRISPR nuclease. For example, the type II CRISPR nuclease can be a Cas9 protein. Suitable Cas9 nucleases include Streptococcus pyogenes Cas9 (SpCas9), Francisella novicida Cas9 (FnCas9), Staphylococcus aureus (SaCas9), Streptococcus thermophilus Cas9 (StCas9), Streptococcus pasteurianus (SpaCas9), Campylobacter jejuni Cas9 (CjCas9), Neisseria meningitis Cas9 (NmCas9), or Neisseria cinerea Cas9 (NcCas9). In other embodiments, the CRISPR nuclease may be derived from a type V CRISPR nuclease, such as Cpf1 nuclease. Suitable Cpf1 nucleases include Francisella novicida Cpf1 (FnCpf1), Acidaminococcus sp. Cpf1 (AsCpf1), or Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). In yet another embodiment, the CRISPR nuclease may be derived from a type VI CRISPR nuclease, such as Leptotrichia wadei Cas13a (LwaCas13a) or Leptotrichia shahii Cas13a (LshCas13a).

[0052] CRISPR nuclease can be wild-type CRISPR nuclease, modified CRISPR nuclease, or a fragment of wild-type or modified CRISPR nuclease.CRISPR nuclease can be modified to increase nucleic acid binding affinity and / or specificity, change enzymatic activity, and / or change other properties of protein.For example, the nuclease (i.e., DNase, RNase) domain of CRISPR nuclease can be modified, deleted, or inactivated.CRISPR nuclease can be truncated to remove domains that are not essential for nuclease function.

[0053] CRISPR nuclease comprises two nuclease domains.For example, Cas9 nuclease comprises HNH domain that cuts the complementary strand of guide RNA and RuvC domain that cuts non-complementary strand, Cpf1 nuclease comprises RuvC domain and NUC domain, and Cas13a nuclease comprises two HNEPN domains.When both nuclease domains function, CRISPR nuclease introduces double-strand break.Either nuclease domain can be inactivated by one or more mutations and / or deletions, thereby creating a variant that introduces single-strand break in one strand of double-stranded sequence. For example, one or more mutations in the RuvC domain of Cas9 nuclease (e.g., D10A, D8A, E762A, and / or D986A) result in an HNH nickase that nicks the complementary strand of the guide RNA; and one or more mutations in the HNH domain of Cas9 nuclease (e.g., H840A, H559A, N854A, N856A, and / or N863A) result in a RuvC nickase that nicks the non-complementary strand of the guide RNA. Comparable mutations can turn Cpf1 and Cas13a nucleases into nickases. Two CRISPR nickases that target opposing strands of a chromosomal sequence (through a pair of offset guide RNAs) can be used in combination to generate double-strand breaks in the chromosomal sequence. Dual CRISPR nickase RNPs can improve target specificity and reduce off-target effects.

[0054] Additional Domains The CRISPR nuclease may further comprise at least one nuclear localization sequence (NLS). NLS is an amino acid sequence that targets zinc finger nuclease proteins into the nucleus and facilitates the introduction of double-strand breaks in target sequences in chromosomes. Nuclear localization signals are known in the art (see, for example, Lange et al., J. Biol. Chem., 2007, 282:5101-5105). Non-limiting examples of nuclear localization signals include PKKKRKV (SEQ ID NO: 1), PKKKRRV (SEQ ID NO: 2), KRPAATKKAGQAKKKK (SEQ ID NO: 3), YGRKKRRQRRR (SEQ ID NO: 4), RKKRRQRRR (SEQ ID NO: 5), PAAKRVKLD (SEQ ID NO: 6), RQRRNELKRSP (SEQ ID NO: 7), VSRKRPRP (SEQ ID NO: 8), PPKKARED (SEQ ID NO: 9), PQPKKKPL (SEQ ID NO: 10), SALIKKKKKKMAP (SEQ ID NO: 11), PKQKKRK (SEQ ID NO: 12), RKLKKKIKKL (SEQ ID NO: 13), REKKKFLKRR (SEQ ID NO: 14), KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15), RKCLQAGMNLEARKTKK (SEQ ID NO: 16), NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 17), and RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 18). The NLS can be located at the N-terminus, C-terminus, or internally of the CRISPR nuclease.

[0055] In further embodiments, CRISPR nuclease can also include at least one cell membrane permeation domain.Examples of suitable cell membrane permeation domain include, but are not limited to, GRKKRRQRRRPPQPKKKRKV (SEQ ID NO: 19), PLSSIFSRIGDPPKKKRKV (SEQ ID NO: 20), GALFLGWLGAAGSTMGAPKKKRKV (SEQ ID NO: 21), GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 22), KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 23), YARAAARQARA (SEQ ID NO: 24), THLPRRRRR (SEQ ID NO: 25), GGRRRRR (SEQ ID NO: 26), RRQRRRTSKLMKR (SEQ ID NO: 27), GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 28), KALAWEAKLAKALAKHLAKALAKALKCEA (SEQ ID NO: 29) and RQIKIWFQNRRMKWKK (SEQ ID NO: 30). The cell membrane permeation domain can be located at the N-terminus, C-terminus, or internally of the CRISPR protein.

[0056] In yet other embodiments, the CRISPR nuclease may further comprise at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags. In certain embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato), or any other suitable fluorescent protein. In another embodiment, the marker domain may be a purification tag and / or an epitope tag.Suitable tags include, but are not limited to, poly(His) tag, FLAG (or DDK) tag, Halo tag, AcV5 tag, AU1 tag, AU5 tag, biotin carboxyl carrier protein (BCCP), calmodulin binding protein (CBP), chitin binding domain (CBD), E tag, E2 tag, ECS tag, eXact tag, Glu-Glu tag, glutathione-S-transferase (GST), HA tag, HSV tag, KT3 tag, maltose binding protein (MBP), MAP tag, Myc tag, NE tag, NusA tag, PDZ tag, S tag, S1 tag, SBP tag, Softag 1 tag, Softag 3 tag, Spot tag, Strep tag, SUMO tag, T7 tag, tandem affinity purification (TAP) tag, thioredoxin (TRX), V5 tag, VSV-G tag, and Xa tag. The marker domain can be located at the N-terminus, C-terminus, or internally of the CRISPR nuclease.

[0057] At least one nuclear localization signal, at least one cell membrane permeable domain, and / or at least one marker domain may be directly linked to the CRISPR nuclease via one or more chemical bonds (e.g., covalent bonds). Alternatively, at least one nuclear localization signal, at least one cell membrane permeable domain, and / or at least one marker domain may be indirectly linked to the CRISPR nuclease via one or more linkers. Suitable linkers include amino acids, peptides, nucleotides, nucleic acids, organic linker molecules (e.g., maleimide derivatives, N-ethoxybenzylimidazole, biphenyl-3,4',5-tricarboxylic acid, p-aminobenzyloxycarbonyl, etc.), disulfide linkers, and polymer linkers (e.g., PEG). Linkers may include one or more spacer groups, including, but not limited to, alkylene, alkenylene, alkynylene, alkyl, alkenyl, alkynyl, alkoxy, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, etc. The linker may be neutral or may have a positive or negative charge. In addition, the linker may be cleavable such that the covalent bond of the linker that connects the linker to another chemical group may be broken or cleaved under certain conditions, including pH, temperature, salt concentration, light, catalyst, or enzyme. In some embodiments, the linker may be a peptide linker. The peptide linker may be a flexible amino acid linker or a rigid amino acid linker. Further examples of suitable linkers are well known in the art, and programs for designing linkers are readily available in the art.

[0058] Guide RNA CRISPR nucleases are guided to their target sites by guide RNAs, which hybridize to the target site and interact with the CRISPR nuclease, directing it to the target site in the chromosomal sequence. The target site contains protospacer adjacent motifs ( p rotospacer a djacent motif: PAM). CRISPR proteins of different bacterial species recognize different PAM sequences. For example, PAM sequences include 5'-NGG (SpCas9, FnCas9), 5'-NGRRT (SaCas9), 5'-NNAGAAW (StCas9), 5'-NNNNGATT (NmCas9), 5-NNNNRYAC (CjCas9), and 5'-TTTV (Cpf1), where N is defined as any nucleotide, R is defined as either G or A, W is defined as either A or T, Y is defined as either C or T, and V is defined as A, C, or G. Cas9 PAM is located 3' of the target site, and cpf1 PAM is located 5' of the target site.

[0059] Guide RNAs contain three regions: a first region at the 5' end that is complementary to the sequence of the target site, a second internal region that forms a stem-loop structure, and a third 3' region that remains essentially single-stranded. The first region of each guide RNA is different so that each guide RNA directs the CRISPR nuclease to a specific target site. The second and third regions (also called scaffold regions) of each guide RNA can be the same for all guide RNAs.

[0060] The first region of the guide RNA is complementary to the sequence of the target site (i.e., the protospacer sequence) such that the first region of the guide RNA can base pair with the sequence of the target site. The complementarity between the first region of the guide RNA (i.e., the crRNA) and the target sequence can be at least 80%, at least 85%, at least 90%, at least 95%, or more. Generally, there is no mismatch between the first region of the guide RNA and the sequence of the target site (i.e., the complementarity is complete). In various embodiments, the first region of the guide RNA can comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region of base pairing between the first region of the guide RNA and the target site of the chromosomal sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length. In exemplary embodiments, the first region of the guide RNA is about 19, 20, or 21 nucleotides in length.

[0061] The guide RNA also comprises a second region that forms a secondary structure. In some embodiments, the secondary structure comprises a stem (or hairpin) and a loop. The length of the loop and the stem can vary. For example, the loop can range from about 3 to about 10 nucleotides in length, and the stem can range from about 6 to about 20 base pairs in length. The stem can include one or more bulges of 1 to about 10 nucleotides. That is, the length of the entire second region can range from about 16 to 60 nucleotides in length. In an exemplary embodiment, the loop is about 4 nucleotides in length, and the stem comprises about 12 base pairs.

[0062] Guide RNA also comprises a third region at the 3' end, which remains essentially single-stranded.Thus, the third region does not have any complementarity to any chromosomal sequence in the cell of interest, and does not have any complementarity to the rest of the guide RNA.The length of the third region can vary.Generally, the third region is more than about 4 nucleotides in length.For example, the length of the third region can range from about 5 to about 60 nucleotides in length.

[0063] The combined length of the second and third regions (or scaffold) of the guide RNA can range from about 30 to about 120 nucleotides in length. In some embodiments, the combined length of the second and third regions of the guide RNA ranges from about 70 to 100 nucleotides in length.

[0064] In some embodiments, the guide RNA comprises one molecule that includes all three regions. In other embodiments, the guide RNA may comprise two separate molecules. The first RNA molecule may comprise the first (5') region of the guide RNA and half of the "stem" of the second region of the guide RNA. The second RNA molecule may comprise the other half of the "stem" of the second region of the guide RNA and the third region of the guide RNA. Thus, in this embodiment, the first and second RNA molecules each contain a sequence of nucleotides that is complementary to the other. For example, in some embodiments, the first and second RNA molecules each contain a sequence (of about 6 nucleotides to about 20 nucleotides) that base pairs with the sequence of the other to form a functional guide RNA.

[0065] (iii) Other targeting endonucleases In further embodiments, the targeting endonuclease can be a meganuclease. Meganucleases are endodeoxyribonucleases characterized by long recognition sequences, i.e., the recognition sequences generally range from about 12 base pairs to about 40 base pairs. Because of this requirement, the recognition sequences generally occur only once in any genome. Among meganucleases, a family of homing endonucleases named LAGLIDADG has become a valuable tool for genome research and genome engineering (see, for example, Arnould et al., 2011, Protein Eng Des Sel, 24(1-2):27-31). Other suitable meganucleases include I-CreI and I-Dmol. Meganucleases can be targeted to specific chromosomal sequences by modifying their recognition sequences using techniques well known to those skilled in the art.

[0066] In further embodiments, the targeting endonuclease can be a transcription activator-like effector (TALE) nuclease. TALE is a transcription factor of the plant pathogen Xanthomonas that can be easily engineered to bind to new DNA targets. TALE or its truncated version can be linked to the catalytic domain of an endonuclease such as FokI to create a targeting endonuclease called a TALE nuclease or TALEN (Sanjana et al., 2012, Nat Protoc, 7(1):171-192) and Arnould et al., 2011, Protein Engineering, Design & Selection, 24(1-2):27-31).

[0067] In another embodiment, the targeting endonuclease can be a chimeric nuclease. Non-limiting examples of chimeric nucleases include ZF-meganucleases, TAL-meganucleases, Cas9-FokI fusions, ZF-Cas9 fusions, TAL-Cas9 fusions, etc. Those skilled in the art are familiar with the means to generate such chimeric nuclease fusions.

[0068] In yet another embodiment, the targeting endonuclease can be a site-specific endonuclease. In particular, the site-specific endonuclease can be a "rare-cutter" endonuclease whose recognition sequence occurs rarely in genome. Alternatively, the site-specific endonuclease can be engineered to cleave a site of interest (Friedhoff et al., 2007, Methods Mol Biol 352:1110123). In general, the recognition sequence of the site-specific endonuclease occurs only once in genome. In another further embodiment, the targeting endonuclease can be an artificial targeted DNA double strand break inducer.

[0069] (b) Delivery of the targeted endonuclease to cells The method includes introducing a targeting endonuclease into a parent cell line of interest. The targeting endonuclease can be introduced into the cell as a purified isolated protein or as a nucleic acid encoding the targeting endonuclease. The nucleic acid can be DNA or RNA. In an embodiment where the encoding nucleic acid is mRNA, the mRNA can be 5' capped and / or 3' polyadenylated. In an embodiment where the encoding nucleic acid is DNA, the DNA can be linear or circular. The nucleic acid can be part of a plasmid or viral vector, where the encoding DNA can be operably linked to a suitable promoter. Those skilled in the art are familiar with suitable vectors, promoters, other control elements, and means of introducing the vector into a cell of interest. In an embodiment where the targeting endonuclease is a CRISPR nuclease, the CRISPR nuclease system can be introduced into the cell as a gRNA-protein complex.

[0070] The targeting endonuclease molecule can be introduced into cells by various means. Suitable delivery means include microinjection, electroporation, sonoporation, microprojectile bombardment, calcium phosphate-mediated transfection, cationic transfection, liposomal transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, nucleic acid uptake enhancement by proprietary agents, and delivery via liposomes, immunoliposomes, virosomes, or artificial virions. In certain embodiments, the targeting endonuclease molecule is introduced into cells by nucleofection.

[0071] Optional Donor Polynucleotide The method for targeted genome modification or manipulation further comprises introducing at least one donor polynucleotide into cells, the donor polynucleotide comprising a sequence having at least one nucleotide change compared to the target chromosomal sequence. The donor polynucleotide has substantial sequence identity to the sequence at or near the target site in the chromosomal sequence, so that the double-strand break introduced by the targeting endonuclease can be repaired by homology-directed repair process, and the sequence of the donor polynucleotide can be inserted or exchanged into the chromosomal sequence, thereby modifying the chromosomal sequence. For example, the donor polynucleotide can comprise a first sequence having substantial sequence identity to the sequence on one side of the target site and a second sequence having substantial sequence identity to the sequence on the other side of the target site. The donor polynucleotide can further comprise a donor sequence for integration into the target chromosomal sequence. For example, the donor sequence can be an exogenous sequence (e.g., a marker sequence) such that the integration of the exogenous sequence disrupts the reading frame and inactivates the target chromosomal sequence.

[0072] The length of the first and second sequences of donor polynucleotides that have substantial sequence identity with the sequence at or near the target site in chromosome sequence can and will vary.Generally, each of the first and second sequences of donor polynucleotides is at least about 10 nucleotides in length.In various embodiments, the donor polynucleotide sequence that has substantial sequence identity with chromosome sequence can be about 15 nucleotides, about 20 nucleotides, about 25 nucleotides, about 30 nucleotides, about 40 nucleotides, about 50 nucleotides, about 100 nucleotides, or more than 100 nucleotides in length.

[0073] The phrase "substantial sequence identity" means that the sequence of the polynucleotide has at least about 75% sequence identity to the chromosomal sequence of interest. In some embodiments, the sequence of the polynucleotide has about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the chromosomal sequence of interest.

[0074] The length of the donor polynucleotide can and will vary. For example, the donor polynucleotide can range from about 20 nucleotides to about 200,000 nucleotides in length. In various embodiments, the donor polynucleotide can range from about 20 nucleotides to about 100 nucleotides in length, from about 100 nucleotides to about 1000 nucleotides in length, from about 1000 nucleotides to about 10,000 nucleotides in length, from about 10,000 nucleotides to about 100,000 nucleotides in length, or from about 100,000 nucleotides to about 200,000 nucleotides in length.

[0075] Typically, the donor polynucleotide is DNA. The DNA can be single-stranded or double-stranded. The DNA can be linear or circular. In some embodiments, the donor polynucleotide can be a single-stranded, linear oligonucleotide containing less than about 200 nucleotides. In other embodiments, the donor polynucleotide can be part of a vector. Suitable vectors include DNA plasmids, viral vectors, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). In still other embodiments, the donor polynucleotide can be a PCR fragment or a nucleic acid complexed to a delivery vehicle such as a liposome or poloxamer.

[0076] The donor polynucleotide can be introduced into the cell simultaneously with the targeting endonuclease molecule. Alternatively, the donor polynucleotide and the targeting endonuclease molecule can be introduced into the cell sequentially. The ratio of the targeting endonuclease molecule to the donor polynucleotide can and will vary. In general, the ratio of the targeting endonuclease molecule to the donor polynucleotide ranges from about 1:10 to about 10:1. In various embodiments, the ratio of the targeting endonuclease molecule to the polynucleotide can be about 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ratio is about 1:1.

[0077] (c) Cell culture The method further comprises maintaining the cell under suitable conditions such that the double-strand break introduced by the targeting endonuclease can be repaired (i) by a non-homologous end joining repair process, such that the chromosomal sequence is modified by deletion, insertion, and / or substitution of at least one nucleotide, or optionally (ii) by a homology-directed repair process, such that the chromosomal sequence is exchanged with the sequence of the polynucleotide, such that the chromosomal sequence is modified. In an embodiment in which a nucleic acid encoding the targeting endonuclease is introduced into the cell, the method comprises maintaining the cell under suitable conditions such that the cell expresses the targeting endonuclease.

[0078] Generally, cells are maintained under conditions suitable for cell growth and / or maintenance. Suitable cell culture conditions are well known in the art and are described, for example, in Santiago et al. (2008) PNAS 105:5809-5814; Moehle et al. (2007) PNAS 104:3055-3060; Urnov et al. (2005) Nature 435:646-651; and Lombardo et al. (2007) Nat. Biotechnology 25:1298-1306. Those skilled in the art understand that cell culture methods are known in the art and can and will vary depending on cell type. In all cases, routine optimization may be used to determine the optimal approach for a particular cell type.

[0079] During this step of the process, the targeting endonuclease recognizes and binds to the targeted cleavage site in the chromosomal sequence and generates a double-strand break, and in the process of repairing the double-strand break, at least one nucleotide deletion, insertion, and / or substitution is introduced into the targeted chromosomal sequence.In certain embodiments, the targeted chromosomal sequence is inactivated.

[0080] Upon confirmation that the chromosomal sequence of interest has been modified, a single clone can be isolated and genotyped (via DNA sequencing and / or protein analysis). Cells containing one modified chromosomal sequence can be subjected to one or more additional rounds of targeted genome modification to modify additional chromosomal sequences, thereby creating double knockouts, triple knockouts, etc.

[0081] (IV) Production of recombinant proteins Another aspect of the present disclosure encompasses a method for producing a recombinant protein in a biological production system. Suitable recombinant proteins are described in section (I)(c). The method includes expressing a recombinant protein of interest in any of the engineered cell lines described in section (I) above, and purifying the expressed recombinant protein. Means for producing or manufacturing recombinant proteins are well known in the art (see, e.g., "Biopharmaceutical Production Technology", Subramanian (ed), 2012, Wiley-VCH; ISBN: 978-3-527-33029-4).

[0082] Recombinant proteins can be purified via a process that includes a clarification step, such as, for example, filtration, and one or more chromatography steps, such as, for example, affinity chromatography, Protein A (or G) chromatography, ion exchange (i.e., cation and / or anion) chromatography.

[0083] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs. The following references provide those skilled in the art with the general definitions of many of the terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless otherwise specified.

[0084] When describing elements of the disclosure or preferred embodiments thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0085] As used herein, the term "endogenous sequence" refers to a chromosomal sequence that is native to the cell.

[0086] The term "exogenous sequence" refers to a chromosomal sequence that is not native to the cell or that has been moved to a different chromosomal location.

[0087] An "engineered" or "genetically modified" cell refers to a cell whose genome has been modified or engineered, i.e., the cell contains at least one chromosomal sequence that has been engineered to contain an insertion of at least one nucleotide, a deletion of at least one nucleotide, and / or a substitution of at least one nucleotide.

[0088] The terms "genome modification" and "genome editing" refer to a process in which a particular endogenous chromosomal sequence is altered such that the chromosomal sequence is modified. The chromosomal sequence may be modified to include at least one nucleotide insertion, at least one nucleotide deletion, and / or at least one nucleotide substitution. The modified chromosomal sequence is inactivated so that no product is produced. Alternatively, the chromosomal sequence may be modified so that an altered product is produced.

[0089] As used herein, "gene" refers to a DNA region (including exons and introns) that codes for a gene product, as well as a DNA region that regulates the production of the gene product, whether or not such regulatory sequences are adjacent to the coding sequence and / or the sequence to be transcribed.Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus control regions.

[0090] The term "heterologous" refers to material that is not native to the cell or species of interest.

[0091] The terms "nucleic acid" and "polynucleotide" refer to a polymer of deoxyribonucleotides or ribonucleotides in a linear or cyclic conformation. In this disclosure, these terms should not be interpreted as a limitation on the length of the polymer. These terms can include known analogs of natural nucleotides, as well as nucleotides modified in the base, sugar, and / or phosphate moieties. In general, an analog of a particular nucleotide has the same base-pairing specificity; that is, an analog of A base pairs with T. The nucleotides of a nucleic acid or polynucleotide may be linked by phosphodiester bonds, phosphothioate bonds, phosphoramidate bonds, phosphorodiamidate bonds, or combinations thereof.

[0092] The term "nucleotide" refers to deoxyribonucleotides or ribonucleotides. A nucleotide may be a standard nucleotide (i.e., adenosine, guanosine, cytidine, thymidine, and uridine) or a nucleotide analog. A nucleotide analog refers to a nucleotide with a modified purine or pyrimidine base, or a modified ribose moiety. A nucleotide analog may be a naturally occurring nucleotide (e.g., inosine) or a non-naturally occurring nucleotide. Non-limiting examples of modifications to the sugar or base moiety of a nucleotide include the addition (or removal) of acetyl, amino, carboxyl, carboxymethyl, hydroxy, methyl, phosphoryl, and thiol groups, and the replacement of carbon and nitrogen atoms of the base with other atoms (e.g., 7-deazapurines). Nucleotide analogs also include dideoxynucleotides, 2'-O-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.

[0093] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues.

[0094] The term "target site" or "target sequence" as used herein refers to a nucleic acid sequence that defines a portion of a chromosomal sequence to be modified or edited and that is engineered for a targeting endonuclease to recognize and bind to, provided sufficient conditions for binding exist.

[0095] The terms "upstream" and "downstream" refer to positions relative to a fixed position in a nucleic acid sequence, with upstream referring to the region that is 5' to that position (i.e., closer to the 5' end of the strand) and downstream referring to the region that is 3' to that position (i.e., closer to the 3' end of the strand).

[0096] Techniques for determining the identity of nucleic acid and amino acid sequences are well known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA of a gene and / or determining the amino acid sequence encoded thereby, and comparing these sequences with a second nucleotide or amino acid sequence. Genomic sequences can also be determined and compared in this manner. In general, identity refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between the two aligned sequences divided by the length of the shorter sequence, multiplied by 100. Approximate alignment of nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences using a score matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, MO Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, DC, USA, and can be normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm for determining percent identity of sequences is provided by Genetics Computer Group (Madison, Wis.) in a utility application called "BestFit". Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the GenBank website. For the sequences described herein, the desired degree of sequence identity ranges from approximately 80% to 100% and any integer value therebetween. Typically, the percent identity between sequences is at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, even more preferably 95%, and most preferably 98% sequence identity.

[0097] It is intended that all matter contained in the above description and the following examples be interpreted as illustrative and not limiting, as various changes may be made in the above cells and methods without departing from the scope of the invention. EXAMPLES

[0098] The following examples illustrate certain aspects of the invention.

[0099] Example 1: Design of an asparagine-mediated selection system To develop an asparagine-mediated metabolic selection system, we first developed a CHO cell line that is auxotrophic for the non-essential amino acid asparagine (Asn). A comprehensive search to identify all genes related to asparagine synthesis was performed against the Reactome and KEGG databases. We identified the endogenous glutamine hydrolyzing asparagine synthetase gene (ASNS) as the only non-redundant gene responsible for Asn synthesis (Figure 1). To generate the Asn-auxotrophic CHO cell line, we used the glutamine (Gln) auxotroph, CHOZN from MilliporeSigma. (登録商標) GS - / - Cell line (CHOZN (登録商標) ) was used. (登録商標) Through whole genome sequencing (WGS) of the cell lines we revealed the endogenous ASNS coding sequence (Figure 2), which, as determined by digital droplet PCR (ddPCR) analysis, is CHOZN (登録商標) It was found to be present in two copies of the genome of CHOZN (Figure 3). A zinc finger nuclease (ZFN) was designed to disrupt the 10th exon of the ASNS gene, which encodes the active subunit of the ASNS protein. The ZFN target sequence is underlined in Figure 2. (登録商標) Cells were cultured in EX-CELL culture medium supplemented with 6 mM L-glutamine (MilliporeSigma G7513). (登録商標) CD CHO cells were cultured in Fusion medium (MilliporeSigma 14365C) ​​(Fusion+Gln) at 37°C with 5% CO2 under shaking conditions. 0.5e6 cells were seeded the day before transfection and cultures were maintained in logarithmic growth phase. 1.0e6 cells were transfected with 6μg of mRNA for each ZFN arm using electroporation. ZFN cleavage activity was assessed via surveyor nuclease S mutation detection assay (Figure 4). Transfected cells were cultured in EX-CELL 2000 ng / ml medium supplemented with 6mM L-glutamine (MilliporeSigma G7513) and 2mM L-anhydrous asparagine (MilliporeSigma 1043502). (登録商標)The cells were transferred to a 6-well culture flask containing 3 mL of CD CHO Fusion medium (MilliporeSigma 14365C) ​​(Fusion+Gln+Asn). The cells were incubated at 30° C. and 5% CO2 in a static environment for 48 hours, after which they were transferred to a 37° C. / 5% CO2 static environment for the next 48 hours. 96 hours after transfection, the ZFN-modified CHOZNs were (登録商標) Cells were scaled up into T-75 flasks. Single-cell clones were isolated from the ZFN-modified pools into 96-well culture plates via a fluorescence-activated cell sorter (FACS). Single-cell clones were assessed via next-generation sequencing (NGS) to identify clones with successful genetic disruption of both copies of ASNS (Figure 5).

[0100] To demonstrate the effectiveness of the asparagine-mediated selection mechanism both as a stand-alone system and as part of a dual metabolic selection system, we generated stable selected cell populations expressing multiple molecules. The molecules used to validate this system included Capsicum red fluorescent protein (RFP), Dasher green fluorescent protein (GFP), and human IgG1. (登録商標) GS - / - ASNS - / - Cells were cultured in Fusion+Gln+Asn medium. Expression vectors used in this study contain either asparagine synthetase (ASNS) or glutamine synthetase (GS) selection markers as specified by the experimental design. Expression of mouse ASNS (protein: asparagine synthetase {glutamine hydrolysis}; gene: ASNS; UniProtKB ID: Q61024) or mouse GS (protein: glutamine synthetase {glutamate ammonia ligase}; gene: Glul; UniProtKB ID: P15105) was driven by the 5' endogenous expression control sequence (promoter) or the 5' SV40 promoter with the SV40 polyadenylation sequence at the 3' end of the gene (Figures 6 and 7).

[0101] CHOZN (登録商標) GS - / - ASNS- / - Cells were cultured in Fusion+Gln+Asn under shaking conditions at 37°C and 5% CO2. 0.5e6 cells were seeded the day before transfection and cultures were maintained in logarithmic growth phase. 1.0e6 cells per condition were transfected with 12μg of plasmid DNA using electroporation. Transfected cells were transferred to T-25 culture flasks containing Fusion+Gln+Asn and 2mL of medium was added 48 hours post-transfection. 96 hours post-transfection, cells were pelleted, medium aspirated and then resuspended in 12mL of appropriate selection medium at 4e5 viable cells / mL and transferred to T-75 flasks. Glutamine-based selection was performed using Fusion-Gln, asparagine-based selection was performed using Fusion-Asn, and glutamine / asparagine double selection was performed using Fusion with low glutamine and -Asn. The cell viability and viable cell density of the various selected cultures were monitored over time. Upon recovery from selection, stable selected cultures were transferred to TPP for scale-up and adaptation to shaking conditions. (登録商標) TubeSpin Bioreactor Tubes (TPP (登録商標) ) was moved to.

[0102] EX-CELL (登録商標) Advanced CHO Fed-batch medium (MilliporeSigma 14366C), EX-CELL (登録商標) Advanced CHO Feed (MilliporeSigma 24367C), and Cellvento (登録商標)Custom formulations of 4 Feed (MilliporeSigma 1.03796.0005) lacking L-anhydroasparagine (Asn) were developed (Advanced-Asn, Feed-Asn, and 4 Feed-Asn, respectively). Stable selected cultures transfected with an IgG1 expression vector were pelleted, the selection medium aspirated, and then resuspended at 3e5 viable cells / mL in Advanced-Asn for productivity analysis in fed-batch conditions. Viable cell density and viability of each culture were collected every other day starting on day 3 after seeding. Starting on day 3 after seeding, 1.5 mL of a 50:50 mixture of Advanced Feed -Asn and 4 Feed -Asn was added to each culture. Glucose and glutamine measurements were obtained from each culture every other day from day 5, and D-glucose (MilliporeSigma G8769) and L-glutamine (MilliporeSigma G7513) were added to maintain adequate glucose and glutamine levels. Productivity was monitored over time and fed batch titers were recorded every other day from day 9 until culture viability fell below 70%. Titers were determined using interferometry on a ForteBio Octet and then confirmed via HPLC Protein A affinity chromatography.

[0103] Example 2: Based on EX-CELL (登録商標) A custom formulation of CD CHO Fusion medium was developed that does not contain L-anhydroasparagine (Asn) (Fusion-Gln-Asn). (登録商標) GS - / - ASNS - / - Clones were cultured in Fusion+Gln+Asn or Fusion+Gln-Asn for at least 10 days. Viability and viable cell density measurements were performed twice weekly. (登録商標) GS - / - ASNS - / - cannot grow in the absence of asparagine, but CHOZN can grow when asparagine is added to the medium. (登録商標) GS - / - ASNS- / - It is shown that cell proliferation is rescued.

[0104] Example 3: To demonstrate that the stable selected cell populations were capable of producing the protein of interest using the Asn-mediated selection system described in Example 1, cells were transfected and subcultured under selection pressure in Fusion-Asn. The ASNS / RFP vector was transfected into CHOZN (登録商標) GS - / - ASNS - / - Cells were transfected with ASNS / RFP plasmids. Cell growth and viability were monitored throughout selection. Cells transfected with ASNS / RFP plasmids and cultured in either Fusion+Gln+Asn (non-selective medium) or Fusion+Gln-Asn (Asn-selective medium) were allowed to recover from selection pressure. Viable cell populations were analyzed by FACS, and mean fluorescence intensity (MFI) and percentage of RFP+ cells were measured. Cells grown in Fusion+Gln+Asn showed a lower percentage of RFP-positive cells and lower MFI compared to cells that underwent Asn-selection in Fusion+Gln-Asn. The results are summarized in Table 1.

[0105] [Table 1]

[0106] Example 4: To demonstrate that a stable selected cell population can produce a protein of interest using the Asn-mediated selection system described in Example 1, a vector was developed with coding sequences for IgG heavy chain, IgG light chain, and asparagine synthetase (ASNS) driven by either the 5' endogenous expression control sequence or the 5' SV40 promoter (Figures 6 and 7). (登録商標) GS - / - ASNS - / -The cell lines were transfected with Advanced Feed -Asn and 4 Feed -Asn. As a control, mock transfections without DNA were used. The populations were subcultured under selection pressure in Fusion+Gln-Asn. The conditions used for selection were also applied during recovery, scale-up, and productivity assays. 3e5 viable cells / mL in Advanced+Gln-Asn medium were inoculated into the Fed-Batch productivity assay. Viable cell density and viability of each culture were collected every other day from day 3 after seeding. From day 3 after seeding, 1.5 mL of a 50:50 mixture of Advanced Feed -Asn and 4 Feed -Asn were added to each culture. Glucose and glutamine measurements were obtained every other day from day 5, and D-+-glucose (MilliporeSigma G8769) and L-glutamine (MilliporeSigma G7513) were added to maintain adequate levels of glucose and glutamine. Cells selected with the ASNS coding sequence driven by the endogenous expression control sequence (ASNS promoter) were shown to exhibit approximately three-fold higher IgG expression levels than cells expressing the same IgG when selected with the ASNS coding sequence driven by the SV40 promoter (Figure 9).

[0107] Example 5: To test whether the stable cell populations could produce two independent intracellular fluorescent proteins under double selection conditions, two vectors were developed, the first containing sequences encoding GFP and GS, and the second containing sequences encoding RFP and ASNS (Figure 6). These two plasmids were transformed into CHOZN (登録商標) GS - / - ASNS - / - As a control, each vector was co-transfected with CHOZN (登録商標) GS - / - ASNS - / -Cells were transfected separately (GFP only and RFP only, respectively). Cells from all three transfections were then subcultured under GS selection conditions (Fusion-Gln), ASNS selection conditions (Fusion-Asn), and dual metabolic selection conditions (Fusion-Gln-Asn with amino acids added as required). The conditions used for selection were also applied during recovery, scale-up, and all other assays. Figure 10 shows the growth and viability data from the selection assays, suggesting that cells transfected with the GFP vector can survive and grow in -Gln conditions but require the addition of Asn to the medium. On the other hand, cells transfected with the RFP vector can survive and grow under -Asn conditions but require the addition of Gln to the medium. Cells co-transfected with both vectors (GFP+RFP) can survive and grow in -Gln medium, -Asn medium, and -Gln-Asn medium (with amino acids added as required). 11 and 12 suggest that cells transfected with the GFP vector surviving and growing in -Gln medium are GFP positive, cells transfected with the RFP vector surviving and growing in -Asn medium are RFP positive, and cells co-transfected with both vectors (GFP+RFP) surviving and growing in -Gln-Asn or -Asn medium supplemented with various levels of Gln and / or Asn are positive for both GFP and RFP. This data suggests that the GS+ASNS dual metabolic selection system does not require the addition of any selection substances, such as antibiotics, to the medium and provides a unique opportunity to select cells into which multiple independent vectors encoding intracellular proteins have been introduced.

[0108] Example 6: To test whether the stable cell populations could produce secreted proteins under double selection conditions, two vectors expressing IgG1 were developed, the first containing sequences encoding IgG heavy chain, IgG light chain, and GS, and the second containing sequences encoding the same IgG heavy chain, IgG light chain, and ASNS (Figure 6). These two independent vectors were transformed into CHOZN (登録商標) GS - / - ASNS - / - As a control, each vector was co-transfected into CHOZN cells (GS+ASNS). (登録商標) GS - / - ASNS - / -The cells were then subcultured under selection pressure in medium supplemented with Fusion-Gln (cells transfected with GS only), Fusion-Asn (cells transfected with ASNS only), or Fusion-Gln-Asn (cells transfected with GS+ASNS) or trace amounts of Gln and / or Asn. The conditions used for selection were also applied during recovery, scale-up, and productivity assays. The GS-only selection culture fully recovered after 14 days, while the ASNS-only and GS+ASNS double selection cultures showed similar selection recovery profiles and required 21 days for full recovery. In fed-batch assays, GS-only and ASNS-only cells produced IgG (Figure 13). This suggests that exogenous GS and ASNS produced by the cells, combined with the addition of Gln and Asn as necessary, is sufficient to sustainably produce secreted proteins without slowing cell growth and proliferation as a result of elevated metabolic load. This provides the possibility to run large-scale production bioreactors under dual selection conditions. Doing so using antibiotic selection methods would be difficult due to either the need to separate or purify the antibiotic from the desired secreted protein or the cost of adding antibiotics to large-scale bioreactors. Furthermore, this dual metabolic selection system (GS+ASNS) provides the opportunity to more efficiently select cells into which multiple large vectors (e.g., expressing bispecific antibodies or other large and / or complex proteins) have been introduced.

Claims

1. 1. A method for producing a recombinant protein product, comprising: (a) providing a mammalian cell line engineered to have reduced or abolished expression of endogenous asparagine synthetase (ASNS); (b) introducing a polynucleotide into said mammalian cell line, wherein the polynucleotide encodes a functional ASNS gene and a recombinant protein; (c) culturing the cell line; and (d) purifying the recombinant protein to form a recombinant protein product. A method comprising:

2. 2. The method of claim 1, wherein the mammalian cell line of (a) further comprises reduced or eliminated expression of endogenous glutamine synthetase (GS).

3. 2. The method of claim 1, wherein endogenous ASNS expression is reduced or eliminated by inactivation of the endogenous ASNS gene in a mammalian cell line.

4. 2. The method of claim 1, wherein the endogenous ASNS gene is inactivated using a targeted endonuclease-mediated genome modification technique.

5. 5. The method of claim 4, wherein the targeting endonuclease is a CRISPR ribonucleoprotein complex or a zinc finger nuclease pair.

6. 2. The method of claim 1, wherein the mammalian cell line is a Chinese hamster ovary (CHO) cell line, a baby hamster kidney (BHK) cell line, an NS0 mouse myeloma cell line, an HEK293 cell line, or a Vero African green monkey kidney cell line.

7. 2. The method of claim 1, wherein the cell line is a CHO cell line.

8. 10. The method of claim 1, wherein the recombinant protein product is selected from an antibody, an antibody fragment, a vaccine, a growth factor, a cytokine, a hormone, or a clotting factor.

9. The method of claim 8, wherein the antibody is a bispecific or multispecific antibody.

10. A genetically engineered mammalian cell line for use in a biological production system that has been engineered to have reduced or eliminated expression of endogenous ASNS.

11. 11. The mammalian cell line of claim 10, wherein expression of ASNS is reduced or eliminated through inactivation of at least one allele of the chromosomal sequence encoding ASNS.

12. 11. The mammalian cell line of claim 10, which has been engineered to have reduced or eliminated expression of endogenous glutamine synthetase (GS).

13. The mammalian cell line of claim 11, wherein the chromosomal sequence has been inactivated using a targeted endonuclease-mediated genome modification approach.

14. 14. The mammalian cell line of claim 13, wherein the cell line is a CHO cell line.

15. 11. The mammalian cell line of claim 10, further comprising at least one nucleic acid encoding a recombinant protein selected from an antibody, an antibody fragment, a vaccine, a growth factor, a cytokine, a hormone, or a clotting factor.

16. The mammalian cell line of claim 15, wherein the antibody is a bispecific or multispecific antibody.

17. A polynucleotide comprising a nucleic acid sequence encoding a functional ASNS and at least one recombinant protein of interest.

18. a) a nucleic acid sequence encoding a functional ASNS; b) a nucleic acid sequence encoding a functional GS; and c) a nucleic acid sequence encoding the recombinant protein of interest; A polynucleotide comprising:

19. The method described in claim 1, wherein the functional ASNS gene comprises a heterologous promoter sequence.

20. The method of claim 1, wherein the functional ASNS gene comprises an endogenous promoter sequence.

21. 21. The method of claim 20, wherein the endogenous promoter is the endogenous ASNS promoter.

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