US 12,416,015 B2
Multiplex production and barcoding of genetically engineered cells
Kevin Roy, Mountain View, CA (US); Justin D. Smith, Redwood City, CA (US); Robert P. St. Onge, San Francisco, CA (US); Lars M. Steinmetz, Stanford, CA (US); and James E. Haber, Waltham, MA (US)
Assigned to THE BOARD OF TRUSTEES OF THE LELAND STANFORD JUNIOR UNIVERSITY, Palo Alto, CA (US); and BRANDEIS UNIVERSITY, Waltham, MA (US)
Appl. No. 16/646,999
Filed by The Board of Trustees of the Leland Stanford Junior University, Palo Alto, CA (US); and Brandeis University, Waltham, MA (US)
PCT Filed Sep. 14, 2018, PCT No. PCT/US2018/051240
§ 371(c)(1), (2) Date Mar. 12, 2020,
PCT Pub. No. WO2019/055878, PCT Pub. Date Mar. 21, 2019.
Claims priority of provisional application 62/559,493, filed on Sep. 15, 2017.
Prior Publication US 2020/0270632 A1, Aug. 27, 2020
Int. Cl. C12N 15/85 (2006.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/113 (2010.01); C12N 15/81 (2006.01)
CPC C12N 15/85 (2013.01) [C12N 9/22 (2013.01); C12N 15/1082 (2013.01); C12N 15/113 (2013.01); C12N 15/81 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01)] 15 Claims
 
1. A method for localizing a donor polynucleotide to a genomic target locus in a target cell, the method comprising:
(a) transfecting a target cell with a first recombinant polynucleotide, the first recombinant polynucleotide comprising a genome editing cassette comprising:
(i) a promoter operably linked to a nucleic acid sequence encoding a guide RNA (gRNA) capable of hybridizing at a genomic target locus to be modified, and
(ii) a donor polynucleotide comprising a sequence to be integrated into the genomic target locus;
(b) transfecting the target cell with a second recombinant polynucleotide that encodes a first polypeptide and encodes a second polypeptide, wherein:
(i) the first polypeptide is an RNA-guided nuclease, wherein the RNA-guided nuclease when complexed with the gRNA recognizes and creates a DNA break at the genomic target locus; and
(ii) the second polypeptide is a donor recruitment protein, wherein the donor recruitment protein comprises:
(1) a DNA binding domain that binds to the donor polynucleotide, and
(2) a DNA break site localizing domain that binds to a region near the DNA break at the genomic target locus;
wherein the donor recruitment protein when bound to both the region near the DNA break site and to the donor polynucleotide selectively recruits the donor polynucleotide to the DNA break site, thereby localizing the donor polynucleotide to the genomic target locus,
wherein the DNA binding domain comprises a LexA DNA binding domain and the DNA break site localizing domain comprises a forkhead-associated (FHA) phosphothreonine-binding domain or comprises a TP53BP1 domain.