# US 2019/0309284 A1 · Methods and kits for dynamic targeted hypermutation

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- Number: US 2019/0309284 A1
- Document ID: US20190309284A1
- Status: Published application
- Kind: A1
- Application number: 16/357,790
- Filed: 2019-03-19
- Published: 2019-10-10
- Earliest claimed: 2018-03-19
- Assignees: Massachusetts Institute of Technology
- Applicants: Massachusetts Institute of Technology
- Inventors: Matthew D. Shoulders; Louis John Papa; Christopher Lawrence Moore
- Web page: https://patentgrep.com/patent/US20190309284A1

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C07K 14/245: Escherichia (G)
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 15/907: {in mammalian cells}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12Y 305/04001: Cytosine deaminase (3.5.4.1)
- C12Y 305/04002: Adenine deaminase (3.5.4.2)
- C12Y 305/04003: Guanine deaminase (3.5.4.3)
- C12Y 305/04004: Adenosine deaminase (3.5.4.4)
- C12Y 305/04005: Cytidine deaminase (3.5.4.5)
- C12Y 305/04006: AMP deaminase (3.5.4.6)
- C12Y 305/04007: ADP deaminase (3.5.4.7)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12Y: ENZYMES

## Abstract

Disclosed herein are methodologies and kits for dynamic targeted hypermutation that harness the enzymatic activity of a polynucleic acid-binding protein fused to a nucleobase-editing enzyme to specifically target mutations across a region of interest. These methodologies and kits facilitate the rapid creation of diverse DNA libraries in vivo or in vitro.

## Claims

### Claim 1

A nucleobase-editing fusion protein comprising a processive polynucleic acid-binding protein fused to a nucleobase-editing enzyme which is capable of altering nucleobases in a pre-existing polynucleic acid sequence.

### Claim 2 (depends on 1)

The nucleobase-editing fusion protein of claim 1, wherein:
the processive polynucleic acid-binding protein of the nucleobase-editing fusion protein comprises the amino acid sequence of an RNA polymerase, a DNA polymerase, a DNA methyltransferase, a DNA glycosylase, or a DNA helicase;
the nucleobase-editing enzyme comprises the amino acid sequence of an Apobec protein, a TadA protein, an AMPD protein, a CDA protein, an ADAT protein, an ADAR protein, or a GDA protein; or
a combination thereof.

### Claim 3 (depends on 1)

The nucleobase-editing fusion protein of claim 1, wherein the processive polynucleic acid-binding protein of the nucleobase-editing fusion protein comprises the amino acid sequence of T7 RNA polymerase or a functional variant thereof.

### Claim 4

(canceled)

### Claim 5 (depends on 1)

The nucleobase-editing fusion protein of claim 1, wherein the nucleobase-editing enzyme comprises the amino acid sequence of an Apobec protein, optionally wherein the Apobec protein is rApobec1 or a functional variant thereof.

### Claim 6

(canceled)

### Claim 7 (depends on 1)

The nucleobase-editing fusion protein of claim 1, wherein the nucleobase-editing enzyme comprises the amino acid sequence of a TadA protein, optionally wherein the TadA protein is E. coli TadA comprising an A106V mutation and/or a D108N mutation or a protein homolog comprising a homologous mutation(s).

### Claim 8

(canceled)

### Claim 9

A method of performing dynamic targeted hypermutation comprising contacting at least one polynucleic acid with at least one non-naturally occurring nucleobase-editing fusion protein, wherein:
a. each of the at least one non-naturally occurring nucleobase-editing fusion proteins comprises a processive polynucleic acid-binding protein fused to a nucleobase-editing enzyme;
b. each of the at least one polynucleic acid comprises a target region; and
c. the contacting of the at least one polynucleic acid with the at least one non-naturally occurring nucleobase-editing fusion protein generates mutations at a rate exceeding background mutation rates only in the target region of the at least one polynucleic acid of (b), wherein the background mutation rate of the at least one polynucleic acid of (b) is determined in the absence of the non-naturally occurring nucleobase-editing fusion protein.

### Claim 10 (depends on 9)

The method of claim 9, wherein:
the processive polynucleic acid-binding protein of the nucleobase-editing fusion protein comprises the amino acid sequence of an RNA polymerase, a DNA polymerase, a DNA methyltransferase, a DNA glycosylase, or a DNA helicase;
the nucleobase-editing enzyme comprises the amino acid sequence of an Apobec protein, a TadA protein, an AMPD protein, a CDA protein, an ADAT protein, an ADAR protein, or a GDA protein; or
a combination thereof.

### Claim 11 (depends on 9)

The method of claim 9, wherein the processive polynucleic acid-binding protein of at least one of the at least one non-naturally occurring nucleobase-editing fusion proteins comprises the amino acid sequence of T7 RNA polymerase or a functional variant thereof.

### Claim 12

(canceled)

### Claim 13 (depends on 9)

The method of claim 9, wherein the nucleobase-editing enzyme of at least one of the at least one non-naturally occurring nucleobase-editing fusion proteins comprises:
the amino acid sequence of an Apobec protein, optionally wherein the Apobec protein is rApobec1 or a functional variant thereof;
the amino acid sequence of a TadA protein, optionally wherein the TadA protein is E. coli TadA comprising an A106V mutation and/or a D108N mutation or a protein homolog comprising a homologous mutation(s); or
a combination thereof.

### Claim at position 14

-16. (canceled)

### Claim 17 (depends on 9)

The method of claim 9, wherein each of the at least one polynucleic acid comprises, from 5′ to 3′: a promoter region that is bound by at least one of the at least one non-naturally occurring nucleobase-editing fusion proteins in a sequence-specific manner; the target region; and a terminator region comprising a terminator array.

### Claim 18 (depends on 17)

The method of claim 17, wherein:
the terminator array comprises four or more terminators, optionally four or more T7 UUCG terminators;
the promoter region of at least one of the at least one polynucleic acids comprises the sequence of SEQ ID NO: 21, and/or SEQ ID NO: 22, SEQ ID NO: 23;
the contacting of the at least one polynucleic acid with the at least one non-naturally occurring nucleobase-editing fusion protein occurs in a living cell; or
a combination thereof.

### Claim at position 17

-20. (canceled)

### Claim 21 (depends on 18)

The method of claim 18, wherein:
at least one of the at least one non-naturally occurring nucleobase-editing fusion proteins is encoded on a plasmid, wherein the plasmid has copy number of less than 10;
at least one of the at least one non-naturally occurring nucleobase-editing fusion proteins is conditionally expressed in the living cell; or
a combination thereof.

### Claim 22

(canceled)

### Claim 23 (depends on 18)

The method of claim 18, wherein the living cell contains a modified genome comprising:
a. an integration of a polynucleic acid sequence encoding for and driving the expression of at least one non-naturally occurring nucleobase-editing fusion protein; and/or
b. an integration of a polynucleic sequence comprising, from 5′ to 3′: a promoter region that is bound by at least one of the at least one non-naturally occurring nucleobase-editing fusion proteins in a sequence-specific manner; the target region; and a terminator region comprising a terminator array.

### Claim 24 (depends on 18)

The method of claim 18, wherein the living cell contains a modified genome and a plasmid that facilitates expression of a T7 inhibitor, optionally wherein the T7 inhibitor is T7 lysozyme, wherein the modified genome of the living cell comprises:
a. an integration of a polynucleic acid sequence encoding for and driving the expression of the non-naturally occurring nucleobase-editing fusion protein, wherein the sequence driving the expression of the fusion protein comprises a sequence bound by LacI repressor that inhibits transcription of the fusion protein when LacI is bound; and/or
b. a deletion of genomic sequence encoding for uracil deglycosylase.

### Claim 25

(canceled)

### Claim 26 (depends on 21)

The method of claim 21, wherein the living cell is treated to increase the expression and/or activity of the uracil deglycoslyase inhibitor, ugi.

### Claim 27

A kit for performing dynamic targeted hypermutation comprising:
a. a polypeptide comprising the amino acid sequence of a non-naturally occurring nucleobase-editing fusion protein comprising a processive polynucleic acid-binding protein fused to a nucleobase-editing enzyme or a polynucleic acid sequence encoding for and driving the expression of said polypeptide; and
b. a polynucleic acid sequence comprising, from 5′ to 3′: a promoter region that is bound by the non-naturally occurring nucleobase-editing fusion protein of (a) in a sequence-specific manner; a cloning site; and a terminator region comprising a terminator array.

### Claim 28

(canceled)

### Claim 29 (depends on 27)

The kit of claim 27, wherein:
the processive polynucleic acid-binding protein of the nucleobase-editing fusion protein comprises the amino acid sequence of an RNA polymerase, a DNA polymerase, a DNA methyltransferase, a DNA glycosylase, or a DNA helicase;
the nucleobase-editing enzyme comprises the amino acid sequence of an Apobec protein, a TadA protein, an AMPD protein, a CDA protein, an ADAT protein, an ADAR protein, or a GDA protein; or
a combination thereof.

### Claim 30 (depends on 27)

The kit of claim 27, wherein the processive polynucleic acid-binding protein of the non-naturally occurring nucleobase-editing fusion protein comprises the amino acid sequence of T7 RNA polymerase or a functional variant thereof.

### Claim 31

(canceled)

### Claim 32 (depends on 27)

The kit of claim 27, wherein the nucleobase-editing enzyme of the nucleobase-editing fusion protein comprises:
the amino acid sequence of an Apobec protein, optionally wherein the Apobec protein is rApobec1 or a functional variant thereof;
the amino acid sequence of a TadA protein, optionally wherein the TadA protein is E. coli TadA comprising an A106V mutation and/or a D108N mutation or a protein homolog comprising a homologous mutation(s); or
a combination thereof.

### Claim at position 30

-35. (canceled)

### Claim 36 (depends on 27)

The kit of claim 27, wherein;
the terminator array comprises four or more terminators, optionally four or more T7 UUCG terminators;
the promoter region comprises the sequence of SEQ ID NO: 21, SEQ ID NO: 22, and/or SEQ ID NO: 23; or
a combination thereof.

### Claim 37

(canceled)