Mammalian cells comprising integrated CAS9 genes to produce stable integration sites, and mammalian cells comprising stable integration sites and other sites
The present inventions provide mammalian cells that comprise multiple Stable Integration Sites. The inventions provide sites introduced genomically into a Genomic Safe Harbor and introduced genomically outside of that particular Genomic Safe Harbor, including but not limited to another Genomic Safe Harbor. Polynucleotides of interest that encode polypeptides or RNAs of interest can be inserted into the Stable Integration Sites provided according to the inventions. The cells and methods of the inventions can be used for the high yield production of any protein, including viral proteins. Additionally, the cells and methods of the inventions are useful for production of viral vectors, such as AAV, antibodies and other proteins.
1 . A mammalian cell comprising a site for integration of a gene of interest, wherein the cell comprises a first Stable Integration Site located in a Genomic Safe Harbor and a second Stable Integration Site that is not located in the Genomic Safe Harbor,
wherein the first Stable Integration Site comprises a DNA polynucleotide comprising in 5′ to 3′ order a first recombinase recognition site (RRS), a first reporter gene encoding a first reporter protein, a Cas9 gene and a second RRS; and wherein the second Stable Integration Site comprises a DNA polynucleotide comprising in 5′ to 3′ order a first Genomic Safe Harbor homology arm containing a CRISPR single guide RNA (sgRNA) target site, a third RRS, a second reporter gene encoding a second reporter protein, a fourth RRS and a second Genomic Safe Harbor homology arm containing an CRISPR sgRNA target site, wherein the first RRS, the second RRS, the third RRS and the fourth RRS are different, and wherein the first reporter protein and the second reporter protein are different.
2 . The mammalian cell according to claim 1 , wherein the second Stable Integration Site is located in a second Genomic Safe Harbor that is different from the first Genomic Safe Harbor.
3 . A mammalian cell comprising a site for integration of a gene of interest, wherein the cell comprises a first Stable Integration Site located in a Genomic Safe Harbor and a second Stable Integration Site that is not located in the Genomic Safe Harbor,
wherein the first Stable Integration Site comprises a first DNA polynucleotide encoding a first reporter protein, wherein the first DNA polynucleotide comprises in 5′ to 3′ order a first recombinase recognition site (RRS), a first reporter gene encoding the first reporter protein, a Cas9gene and a second RRS; and wherein the second Stable Integration Site comprises a second DNA polynucleotide encoding a second reporter protein, wherein the second DNA polynucleotide comprises in 5′ to 3′ order a first Genomic Safe Harbor homology arm containing a CRISPR single guide RNA (sgRNA) target site, a third RRS, a second reporter gene encoding the second reporter protein, a fourth RRS and a second Genomic Safe Harbor homology arm containing an CRISPR sgRNA target site, and wherein the first RRS, the second RRS, the third RRS and the fourth RRS are different.
4 . The mammalian cell according to claim 3 , wherein the second Stable Integration Site is located in a second Genomic Safe Harbor that is different from the first Genomic Safe Harbor.
5 . The mammalian cell according to claim 3 , wherein the mammalian cell is a human cell.
6 . The mammalian cell according to claim 3 , wherein the mammalian cell is selected from the group consisting of a Human Amniotic Epithelial Cell, a HEK293 Cell, a BHK cell, and a CHO cell.
7 . The mammalian cell according to claim 1 , wherein the mammalian cell is selected from the group consisting of a Human Amniotic Epithelial Cell, a HEK293 Cell, a BHK cell, and a CHO cell.
8 . The mammalian cell according to claim 1 , wherein the Cas9 gene can be removed using a recombinase.
9 . The mammalian cell according to claim 1 , wherein the Cas9 gene encodes Cas9 protein, wherein the Cas9 protein is used to produce at least one Stable Integration Site in the Genomic Safe Harbor for stable integration of DNA polynucleotides.
10 . The mammalian cell according to claim 9 , wherein the mammalian cell can have more than one Stable Integration Site for stable integration.
11 . The mammalian cell according to claim 1 , wherein the mammalian cell further comprises a DNA polynucleotide encoding a repressor under the control of a promoter.
12 . The mammalian cell according to claim 11 , wherein the repressor is a Tet repressor.
13 . The mammalian cell according to claim 1 , wherein the mammalian cell comprises a DNA polynucleotide encoding a repressor protein and a polyadenylation signal under transcriptional control of a promoter, wherein the DNA polynucleotide encoding the repressor protein is randomly or site-specifically inserted in the cell genome.
14 . The mammalian cell according to claim 1 , wherein the integrated Cas9 gene is under the control of a promoter.
15 . The mammalian cell according to claim 1 , wherein the expression of Cas9 gene increases the efficiency of homology arm integration into a Genomic Safe Harbor by increasing the occurrence of cuts in genomic DNA caused by Cas9 endonuclease.
16 . The mammalian cell according to claim 9 , wherein the integrated Cas9 gene provides greater homology directed repair efficiency than homology directed repair without an integrated Cas9 gene.
17 . The mammalian cell according to claim 16 , wherein the integrated Cas9 gene provides 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 greater homology directed repair efficiency than homology directed repair without an integrated Cas9 gene.
18 . The mammalian cell according to claim 1 further comprising a CMV promoter operably linked to a Tet operator to control transcription of the Cas9 gene.
19 . The mammalian cell according to claim 1 , wherein the first and second Genomic Safe Harbor homology arms each contain a region with a sequence alteration to avoid recreating a targetable site.
20 . The mammalian cell according to claim 1 , wherein the first and second Genomic Safe Harbor homology arms can target a sequence according to SEQ ID NO:2.
21 . The mammalian cell according to claim 20 , wherein the first and second Genomic Safe Harbor homology arms each contain a region with a sequence alteration to avoid recreating a targetable site.
22 . The mammalian cell according to claim 1 , wherein a gene of interest can be inserted between the third RRS and the fourth RRS.
23 . The mammalian cell according to claim 3 , wherein the Cas9 gene can be removed using a recombinase.
24 . The mammalian cell according to claim 3 , wherein the Cas9 gene encodes Cas9 protein, wherein the Cas9 protein is used to produce at least one Stable Integration Site in the Genomic Safe Harbor for stable integration of DNA polynucleotides.
25 . The mammalian cell according to claim 24 , wherein the mammalian cell can have more than one Stable Integration Site for stable integration.
26 . The mammalian cell according to claim 3 , wherein the mammalian cell further comprises a DNA polynucleotide encoding a repressor under the control of a promoter.
27 . The mammalian cell according to claim 26 , wherein the repressor is a Tet repressor.
28 . The mammalian cell according to claim 3 , wherein the mammalian cell comprises a DNA polynucleotide encoding a repressor protein and a polyadenylation signal under transcriptional control of a promoter, wherein the DNA polynucleotide encoding the repressor protein is randomly or site-specifically inserted in the cell genome.
29 . The mammalian cell according to claim 3 , wherein the integrated Cas9gene is under the control of a promoter.
30 . The mammalian cell according to claim 3 , wherein the expression of Cas 9 gene increases the efficiency of homology arm integration into a Genomic Safe Harbor by increasing the occurrence of cuts in genomic DNA caused by Cas9 endonuclease.
31 . The mammalian cell according to claim 24 , wherein the integrated Cas9gene provides greater homology directed repair efficiency than homology directed repair without an integrated Cas9 gene.
32 . The mammalian cell according to claim 31 , wherein the integrated Cas9gene provides 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 greater homology directed repair efficiency than homology directed repair without an integrated Cas9 gene.
33 . The mammalian cell according to claim 3 further comprising a CMV promoter operably linked to a Tet operator to control transcription of the Cas9 gene.
34 . The mammalian cell according to claim 3 , wherein the first and second Genomic Safe Harbor homology arms each contain a region with a sequence alteration to avoid recreating a targetable site.
35 . The mammalian cell according to claim 3 , wherein the first and second Genomic Safe Harbor homology arms can target a sequence according to SEQ ID NO:2.
36 . The mammalian cell according to claim 35 , wherein the first and second Genomic Safe Harbor homology arms each contain a region with a sequence alteration to avoid recreating a targetable site.
37 . The mammalian cell according to claim 3 , wherein a gene of interest can be inserted between the third RRS and the fourth RRS.