US 12,402,609 B2
Methods for producing hypo-allergenic cats using gene editing technology
David B. Avner, Highlands Ranch, CO (US); Sven Bocklandt, Los Angeles, CA (US); and James Kehler, Silver Spring, MD (US)
Assigned to FELIX PETS, LLC, Highlands Ranch, CO (US)
Appl. No. 16/069,998
Filed by FELIX PETS, LLC, Highlands Ranch, CO (US)
PCT Filed Jan. 13, 2017, PCT No. PCT/US2017/013540
§ 371(c)(1), (2) Date Jul. 13, 2018,
PCT Pub. No. WO2017/124022, PCT Pub. Date Jul. 20, 2017.
Claims priority of provisional application 62/278,293, filed on Jan. 13, 2016.
Prior Publication US 2020/0008405 A1, Jan. 9, 2020
Int. Cl. A01K 67/0275 (2024.01); C12N 5/074 (2010.01); C12N 15/87 (2006.01)
CPC A01K 67/0275 (2013.01) [C12N 5/0696 (2013.01); C12N 15/87 (2013.01); A01K 2217/00 (2013.01); A01K 2227/10 (2013.01); A01K 2267/02 (2013.01)] 17 Claims
 
1. A method of producing a genetically modified cell, the method comprising the steps of:
(a) providing a feline somatic cell, a feline embryonic stem cell, or a feline iPS cell; and
(b) introducing into the cell of (a) two nucleases designed to disrupt a region of the felis domesticus 1 (Fel d I) locus, each nuclease comprising a programmable, sequence-specific, DNA binding module fused to a non-specific DNA cleavage domain; and
(c) screening for a cell in which the region of the Fel d I locus is disrupted;
wherein the nucleases are selected from Cas9, a transcription activator-like (TAL) effector nuclease, and Cpf1;
wherein the region of the Fel d I locus comprises:
the promoter shared by Chain 1 and Chain 2 of the Fel d I locus or a portion thereof;
and at least one of a coding sequence of Chain 1 of the Fel d I locus and a coding sequence of Chain 2 of the Fel d I locus;
wherein when the nuclease is Cas9, one of the two DNA binding modules are designed to target a first recognition site of Fel d I having a sequence selected from SEQ ID NOs: 43, 44, and 45; and the other of the two DNA binding modules are designed to target a second recognition site of Fel d I having a sequence selected from SEQ ID NOs: 46 and 47; and
wherein when the nuclease is TAL, one of the two DNA binding modules is designed to target a recognition site of Fel d I having a sequence selected from SEQ ID NOs: 34, 35, and 36; and the other of the two DNA binding modules is designed to target a recognition site of Fel d I having a sequence selected from SEQ ID NOs: 37, 38, and 39.