Continuous directed evolution

Patent metadata

Field	Value
Publication number	US-12366009-B2
Application number	US-202117541848-A
Country	US
Kind code	B2
Filing date	Dec 3, 2021
Priority date	Dec 22, 2010
Publication date	Jul 22, 2025
Grant date	Jul 22, 2025

Abstract

The invention provides systems, methods, reagents, apparatuses, vectors, and host cells for the continuous evolution of nucleic acids. For example, a lagoon is provided in which a population of viral vectors comprising a gene of interest replicates in a stream of host cells, wherein the viral vectors lack a gene encoding a protein required for the generation of infectious viral particles, and wherein that gene is expressed in the host cells under the control of a conditional promoter, the activity of which depends on a function of the gene of interest to be evolved. Some aspects of this invention provide evolved products obtained from continuous evolution procedures described herein. Kits containing materials for continuous evolution are also provided.

First claim

What is claimed is:

1. A plasmid comprising a conditional promoter linked to a nucleic acid encoding a dominant negative pIII (pIII-neg) protein variant, wherein the conditional promoter comprises a cI-repressed promoter; and wherein activation of the conditional promoter drives pIII-neg expression.

2. The plasmid of claim 1 , wherein the cI-repressed promoter comprises a lambda pR promoter or a lambda pR promoter variant.

3. The plasmid of claim 1 , wherein the cI-repressed promoter comprises one or more lambda repressor (cI) binding sites or one or more cI-neg binding sites.

4. The plasmid of claim 1 , wherein the dominant negative pIII protein variant comprises two N-terminal domains of pIII fused to a truncated, termination-incompetent C-terminal domain of pIII.

5. The plasmid of claim 4 , wherein the C-terminal domain is inactive.

6. An E. coli cell comprising the plasmid of claim 1 .

Assignees

• Harvard College

Inventors

• Liu David R
• Esvelt Kevin Michael
• Carlson Jacob Charles

Classifications

• C12N2795/14152

  relating to complementing cells and packaging systems for producing virus or viral particles · CPC title

• C12N2795/14121

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N15/70

  Vectors or expression systems specially adapted for E. coli · CPC title

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C07K14/005

  from viruses · CPC title