# US 11,299,729 B2 · Vector-based mutagenesis system

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- Number: US 11,299,729 B2
- Document ID: US11299729B2
- Status: Granted
- Kind: B2
- Application number: 15/567,312
- Filed: 2016-04-15
- Published: 2018-03-29
- Granted: 2022-04-12
- Earliest claimed: 2015-04-17
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Ahmed Hussein Badran; David R. Liu
- Web page: https://patentgrep.com/patent/US11299729B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 9/48: acting on peptide bonds (3.4)
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Strategies, reagents, methods, and systems for modulating the mutation rate in cells are provided herein. The strategies, reagents, methods, and systems are broadly applicable for the modulation of mutation rates in cells where high mutation rates and/or control over a broad range of mutation rates is desired, for example, in the context of diversifying a nucleic acid sequence or a plurality of such sequences within a population of cells, for the generation of diversified nucleic acid libraries, and for directed evolution of nucleic acids and encoded products.

## Claims

### Claim 1

An expression construct for modulating the mutation rate of nucleic acids in a bacterial cell, the construct comprising a nucleic acid sequence encoding:
(a) at least one gene product that disrupts a proofreading pathway, a translesion synthesis pathway, and/or a methyl-directed mismatch repair pathway of the bacterial cell; and
(b) at least one gene product that disrupts a base excision repair pathway and/or a base selection pathway of the bacterial cell;
wherein the gene products are under the control of one or more heterologous promoters.

### Claim 2 (depends on 1)

The expression construct of claim 1, wherein the gene product that disrupts a proofreading pathway is a dnaQ926, BRM1, BR11, BR1, BR6, or BR13 gene product.

### Claim 3 (depends on 1)

The expression construct of claim 1, wherein the gene product that disrupts a translesion synthesis pathway is an umuD′, umuC, recA, dinB, or polB gene product.

### Claim 4 (depends on 1)

The expression construct of claim 1, wherein the gene product that disrupts a methyl-directed mismatch repair pathway is a mutS, mutL, mutH, dam, or seqA gene product.

### Claim 5 (depends on 1)

The expression construct of claim 1, wherein the gene product that disrupts a base excision repair pathway is a ugi, AID, APOBEC, CDA, MAG, or AAG gene product.

### Claim 6 (depends on 1)

The expression construct of claim 1, wherein the gene product that disrupts a base selection pathway is a dnaE74, dnaE486, dnaE1026, dnaX36, dnaX2016, emrR, nrdAB, nrdA(H59A)B, nrdA(A65V)B, nrdA(A301V)B, nrdAB(P334L), or nrdEF gene product.

### Claim 7 (depends on 1)

The expression construct of claim 1, wherein the expression construct further comprises a nucleic acid sequence encoding a rsmE, cchA, yffI, or yfjY gene product.

### Claim 8 (depends on 1)

The expression construct of claim 1, wherein the expression construct comprises a nucleic acid sequence encoding a dnaQ926 gene product.

### Claim 9 (depends on 1)

The expression construct of claim 1, wherein the expression construct comprises a nucleic acid sequence encoding a dam gene product.

### Claim 10 (depends on 1)

The expression construct of claim 1, wherein the expression construct comprises a nucleic acid sequence encoding a dnaE74, a dnaE486, a dnaE1026, a dnaX36, a dnaX2016, a rpsD12, a rpsD14, a rpsD16, a polB, a polB(D156A), a MAG1, a AAG(Y127I-H136L), and/or a Δ80-AAG(Y127I-H136L) gene product.

### Claim 11 (depends on 1)

The expression construct of claim 1, wherein the heterologous promoter is an inducible promoter.

### Claim 12 (depends on 1)

The expression construct of claim 1, wherein the expression construct comprises a nucleic acid sequence encoding a: dnaQ926 gene product, a dam gene product, a seqA gene product, an emrR gene product, a ugi gene product, and a CDA1 gene product.

### Claim 13 (depends on 1)

The expression construct of claim 1, wherein the expression construct further comprises:
(i) a nucleic acid sequence encoding pIII protein; and
(ii) a drift promoter;
wherein the nucleic acid sequence encoding pIII protein is under the control of the drift promoter.

### Claim 14 (depends on 1)

A plasmid comprising the expression construct of claim 1.

### Claim 15 (depends on 14)

A cell comprising the plasmid of claim 14.

### Claim 16 (depends on 14)

The plasmid of claim 14, wherein the plasmid comprises a bacterial origin of replication.

### Claim 17 (depends on 16)

The plasmid of claim 16, wherein the origin of replication is a cloDF13 origin of replication.

### Claim 18 (depends on 14)

The plasmid of claim 14, wherein the plasmid comprises a nucleic acid sequence encoding a gene product conferring resistance to an antibiotic to a bacterial host cell.

### Claim 19 (depends on 18)

The plasmid of claim 18, wherein the antibiotic is chloramphenicol.

### Claim 20 (depends on 19)

A method for modulating the mutation rate in a host cell, the method comprising contacting the cell with the plasmid of claim 19.

### Claim 21 (depends on 14)

A method for modulating the mutation rate in a host cell, the method comprising contacting the cell with the plasmid of claim 14.

### Claim 22 (depends on 1)

A cell comprising the expression construct of claim 1.

### Claim 23 (depends on 1)

A method for modulating the mutation rate in a host cell, the method comprising contacting the cell with the expression construct of claim 1.

### Claim 24 (depends on 1)

A method for directed evolution, the method comprising:
(a) contacting a population of host cells comprising the expression construct of claim 1 with a population of phage vectors comprising a gene to be evolved and deficient in at least one gene for the generation of infectious phage particles, wherein
(1) the host cells are amenable to transfer of the vector;
(2) the vector allows for expression of the gene to be evolved in the host cell, can be replicated by the host cell, and the replicated vector can transfer into a second host cell; and
(3) the host cell expresses a gene product encoded by the at least one gene for the generation of infectious phage particles of (a) in response to the activity of the gene to be evolved, and the level of gene product expression depends on the activity of the gene to be evolved;
(b) incubating the population of host cells under conditions allowing for mutation of the gene to be evolved and the transfer of the vector comprising the gene to be evolved from host cell to host cell, wherein host cells are removed from the host cell population, and the population of host cells is replenished with fresh host cells that comprise the expression construct but do not harbor the vector; and
(c) isolating a replicated vector from the host cell population in (b), wherein the replicated vector comprises a mutated version of the gene to be evolved.

### Claim 25 (depends on 1)

A kit comprising:
(a) the expression construct of claim 1, wherein the expression construct comprises an inducible promoter controlling at least one of the nucleic acid sequences comprised in the expression construct; and
(b) an inducing agent that induces expression from the inducible promoter.

### Claim 26

An expression construct comprising a nucleic acid sequence under the control of a heterologous promoter, wherein the expression construct comprises the sequence set forth in any one of SEQ ID NO: 27, 28, 29, 33, 34, and 35.