US 12,291,706 B2
Methods for screening variant of target gene
Alfonso Farruggio, Stanford, CA (US); Ruby Yanru Tsai, San Jose, CA (US); and Lingjie Kong, Union City, CA (US)
Assigned to APPLIED STEMCELL, INC., Milpitas, CA (US)
Appl. No. 16/968,602
Filed by APPLIED STEMCELL, INC., Milpitas, CA (US)
PCT Filed Feb. 8, 2019, PCT No. PCT/US2019/017375
§ 371(c)(1), (2) Date Aug. 9, 2020,
PCT Pub. No. WO2019/157395, PCT Pub. Date Aug. 15, 2019.
Claims priority of provisional application 62/628,236, filed on Feb. 8, 2018.
Prior Publication US 2020/0385710 A1, Dec. 10, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/85 (2006.01); C12N 15/10 (2006.01); C12N 15/90 (2006.01); C40B 40/04 (2006.01)
CPC C12N 15/1082 (2013.01) [C12N 15/1034 (2013.01); C12N 15/907 (2013.01); C12N 2800/30 (2013.01); C12N 2830/002 (2013.01); C12N 2830/003 (2013.01)] 13 Claims
 
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.