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US 12,421,529 B2Granted
Modular genetically engineered cell and methods of generation thereof
AssigneeScience Incorporated
InventorsAlan Mardinly, Kevin Smith, Satoru Miura
Life of this patent
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Abstract
In variants, the method for generating a modular cell can include: integrating a landing site into a cell genome, and integrating a gene of interest into the landing site. The method can optionally include performing a cell selection, removing the gene of interest from the landing site, and/or any other suitable steps.
Claims7
An in vitro method of genetically modifying a cell in multiple loci of the genome, the method comprising:
- (i) transfecting the cell with:
- (a) a first donor vector comprising a first primary landing site sequence and encoding a first gRNA comprising a targeting sequence to a first safe harbor site;
- (b) a second donor vector comprising a second primary landing site sequence and encoding a second gRNA comprising a targeting sequence to a second safe harbor site;
- and
- (c) a CRISPR Cas 9 nuclease;
- wherein the first primary landing site sequence comprises a first pair of site-specific recombinase attachment site sequences flanking a secondary landing site sequence comprising a second pair of site-specific recombinase attachment site sequences, wherein the first pair of site-specific recombinase attachment site sequences are inverted relative to each other, wherein the second primary landing site sequence comprises a third pair of site-specific recombinase attachment site sequences inverted relative to each other, and wherein the first primary landing site sequence integrates into the first safe harbor site in the genome of the cell and the second primary landing site sequence into the second safe harbor site in the genome of the cell;
- and
- (ii) transfecting the cell with:
- (a) a third vector comprising a first gene of interest flanked by a fourth pair of site-specific recombinase attachment sites;
- (b) a fourth vector comprising a second gene of interest flanked by a fifth the pair of site-specific recombinase attachment sites;
- and
- (c) a site-specific recombinase enzyme;
- wherein the fourth pair of site-specific recombinase attachment sites are compatible with the second pair of site-specific recombinase attachment sites, and wherein the first gene of interest integrates into the site of the second pair of site-specific recombinase attachment sites of the secondary landing site sequence;
- and
- wherein the fifth pair of site-specific recombinase attachment sites are compatible with the third pair of site-specific recombinase attachment sites, and wherein the second gene of interest integrates into the site of the second site-specific recombinase attachment sites of the second primary landing site sequence.
12 more elements
Dependencies are inferred from the claim text.