US 12,325,855 B2
Gene editing using homology-independent universal genome engineering technology
Yudong Gao, Durham, NC (US); and Scott Soderling, Durham, NC (US)
Assigned to Duke University, Durham, NC (US)
Appl. No. 16/968,904
Filed by c/o Duke University, Durham, NC (US)
PCT Filed Feb. 15, 2019, PCT No. PCT/US2019/018353
§ 371(c)(1), (2) Date Aug. 11, 2020,
PCT Pub. No. WO2019/161304, PCT Pub. Date Aug. 22, 2019.
Claims priority of provisional application 62/631,360, filed on Feb. 15, 2018.
Prior Publication US 2021/0047643 A1, Feb. 18, 2021
Int. Cl. C12N 15/113 (2010.01); C12N 15/10 (2006.01); C12N 15/63 (2006.01)
CPC C12N 15/113 (2013.01) [C12N 15/102 (2013.01); C12N 15/63 (2013.01); C12N 2310/20 (2017.05)] 15 Claims
 
1. A Homology-Independent Universal Genome Engineering (HiUGE) system for gene editing a subject genome, the HiUGE system comprising:
(a) a Homology-Independent Universal Genome Engineering (HiUGE) vector comprising:
(i) a first polynucleotide sequence encoding at least one insert;
(ii) at least one donor recognition sequence (DRS) flanking each side of the first polynucleotide sequence, the DRS comprising a cleavage site for a CRISPR-based nuclease;
(iii) a second polynucleotide sequence encoding a HiUGE vector specific gRNA, wherein the HiUGE vector specific gRNA targets the CRISPR-based nuclease to the DRS and does not target a specific sequence within the subject genome;
(iv) a third polynucleotide sequence encoding a first portion of a CRISPR-based nuclease having a first split-intein; and
(b) a gene specific vector comprising:
(i) a fourth polynucleotide sequence encoding a second portion of a CRISPR-based nuclease having a second split-intein complementary to the first split-intein, wherein the first portion of a CRISPR-based nuclease and the second portion of a CRISPR-based nuclease can join together to form the CRISPR-based nuclease; and
(ii) a fifth polynucleotide sequence that encodes a target gene specific gRNA which targets the CRISPR-based nuclease to a target gene specific sequence within the subject genome.