US 11,939,571 B2
Library-scale engineering of metabolic pathways
Xiaoge Guo, Brookline, MA (US); Alejandro Chavez, New York, NY (US); Max Schubert, Brookline, MA (US); and Eric Kelsic, Boston, MA (US)
Assigned to President and Fellows of Harvard College, Cambridge, MA (US); and The General Hospital Corporation, Boston, MA (US)
Appl. No. 16/307,998
Filed by President and Fellows of Harvard College, Cambridge, MA (US)
PCT Filed Jun. 12, 2017, PCT No. PCT/US2017/036985
§ 371(c)(1), (2) Date Dec. 7, 2018,
PCT Pub. No. WO2017/214615, PCT Pub. Date Dec. 14, 2017.
Claims priority of provisional application 62/348,438, filed on Jun. 10, 2016.
Prior Publication US 2019/0264196 A1, Aug. 29, 2019
Int. Cl. C12N 15/10 (2006.01); C12N 15/00 (2006.01); C12N 15/86 (2006.01); C12N 15/90 (2006.01); C40B 40/08 (2006.01)
CPC C12N 15/1037 (2013.01) [C12N 15/00 (2013.01); C12N 15/102 (2013.01); C12N 15/1058 (2013.01); C12N 15/86 (2013.01); C12N 15/905 (2013.01); C12N 2310/20 (2017.05); C12N 2800/80 (2013.01); C40B 40/08 (2013.01)] 6 Claims
 
1. A method of generating a library of carrier plasmids in which the carrier plasmids carry a mutation in a nucleic acid segment of a target cell, wherein carrier plasmids within the library carry different mutations in the nucleic acid segment, the method comprising:
synthesizing on an array a library of oligonucleotides, each oligonucleotide comprising:
(a) a segment encoding a guide nucleic acid which is complementary to a target site in a nucleic acid in the target cell, and
(b) a donor DNA comprising the target site which is flanked by two sequences which are complementary to two regions of the nucleic acid of the target cell, wherein the two regions are on opposite sides of the target site, wherein the donor DNA consists of a single nucleotide mutation or more within the target site; and
recombining the library of oligonucleotides with a backbone plasmid,
wherein the oligonucleotides and the backbone plasmid recombine to form a circularized plasmid in which the segment encoding the guide nucleic acid is operably linked downstream from a promoter, wherein the donor DNA in the library of oligonucleotides comprises at least 24 distinct mutations each on a separate molecule of donor DNA.