Methods for screening variant of target gene
Provided are methods for screening a desired variant of a target gene in a eukaryotic system. Compositions for screening a desired variant of a target gene are also provided.
1. A method of generating a mammalian cell library for screening a desired variant of a target gene, the method comprising:
(1) obtaining a first plurality of mammalian cells each of which comprises at genomic locus Hipp11 (H11) an attP site recognized by a Bxb1 integrase;
(2) introducing into the first plurality of mammalian cells a library of nucleic acid constructs wherein at least one of the nucleic acid constructs comprises the desired variant of the target gene, each of the nucleic acid constructs comprising:
a) an attB site recognized by the Bxb1 integrase, and
b) a variant of a target gene,
thereby generating a second plurality of mammalian cells comprising the library of nucleic acid constructs, wherein at least one of the second plurality of mammalian cells comprises the desired variant of the target gene:
(3) expressing in each of the second plurality of mammalian cells the Bxb1 integrase encoded by the polynucleotide sequence of SEQ ID NO: 1 wherein the Bxb1 coding sequence is operably linked to an expression control sequence; and
(4) maintaining the second plurality of mammalian cells under conditions that facilitate recombination between the attP and the attB sites mediated by the Bxb1 integrase to integrate the variants of the target gene at the genomic locus H11, thereby generating the mammalian cell library comprising a third plurality of mammalian cells each of which comprises a variant of the target gene at the genomic locus H11 wherein integration of the variants of the target gene at the genomic locus H11 does not involve a unidirectional recombinase other than the Bxb1 integrase.
2. The method of claim 1 , wherein each of the first plurality of mammalian cells further comprises at the genomic locus H11: a first promoter operably linked to the attP site.
3. The method of claim 2 , wherein the first promoter is a Tet-on promoter.
4. The method of claim 1 , wherein the first plurality of mammalian cells are HEK293 cells.
5. The method of claim 1 , wherein the target gene is an enzyme.
6. The method of claim 1 , wherein the target gene is a second site-specific unidirectional recombinase.
7. The method of claim 6 , wherein the second site-specific unidirectional recombinase is phiC31 integrase.
8. The method of claim 6 , wherein the variant of the second site-specific unidirectional recombinase recognizes a variant unidirectional recombination site.
9. The method of claim 1 , wherein the target gene is a Cas protein.
10. The method of claim 9 , wherein the variant of the Cas protein recognizes a variant protospacer adjacent motif (PAM), or has higher on-target specificity or has lower immunogenicity.
11. The method of claim 1 , wherein the target gene is a virus capsid gene.
12. The method of claim 11 , wherein the virus capsid gene is an AAV capsid gene.
13. The method of claim 11 , wherein the variant of the virus capsid gene has better packaging ability of packaging>4.7kb DNA in size or increased infectivity to a target cell/tissue.