Library-scale engineering of metabolic pathways

Patent metadata

Field	Value
Publication number	US-11939571-B2
Application number	US-201716307998-A
Country	US
Kind code	B2
Filing date	Jun 12, 2017
Priority date	Jun 10, 2016
Publication date	Mar 26, 2024
Grant date	Mar 26, 2024

Abstract

Expressing guide nucleic acids (e.g., gRNA) from the same oligonucleotide that contains donor sequence permits the high efficiency, simultaneous transformation of a population of cells with both substrates. Using oligonucleotide chip array technology, one can construct thousands of oligonucleotides with customized gRNA and donor sequence in a cost effective manner. In combination, one can efficiently modify endogenous and exogenous genes.

First claim

The invention claimed is:

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.

2. The method of claim 1 wherein the circularized plasmid comprises a terminator downstream of the segment encoding the guide nucleic acid.

3. The method of claim 1 wherein the step of recombining is performed in vitro.

4. The method of claim 1 wherein the step of recombining is performed in vivo in cells.

5. The method of claim 1 wherein the backbone plasmid is linearized prior to the step of recombining.

6. The method of claim 1 wherein the circularized plasmid comprises a DNA barcode.

Assignees

• Harvard College
• Massachusetts Gen Hospital

Inventors

• Guo Xiaoge
• Chavez Alejandro
• Schubert Max
• Kelsic Eric

Classifications

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• C12N15/1037 Primary

  Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C12N15/102

  Mutagenizing nucleic acids · CPC title

• C12N15/1058 Primary

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title