# US 2016/0257948 A1 · Methods, Cells & Organisms

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- Number: US 2016/0257948 A1
- Document ID: US20160257948A1
- Status: Published application
- Kind: A1
- Application number: 15/072,794
- Filed: 2016-03-17
- Published: 2016-09-08
- Earliest claimed: 2013-09-18
- Assignees: Kymab Limited
- Applicants: Kymab Limited
- Inventors: Allan Bradley; Hanif Ali; E-Chiang Lee
- Web page: https://patentgrep.com/patent/US20160257948A1

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 5/0635: {B lymphocytes}
- C12N 15/907: {in mammalian cells}
- A01K 2207/15: Humanized animals
- A01K 2217/052: inducing gain of function
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2227/105: Murine
- A01K 2267/01: Animal expressing industrially exogenous proteins
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/20: characterized by taxonomic origin
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C12N 2510/04: Immortalised cells
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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)

## Abstract

The invention relates to an approach for introducing one or more desired insertions and/or deletions of known sizes into one or more predefined locations in a nucleic acid (e.g., in a cell or organism genome). They developed techniques to do this either in a sequential fashion or by inserting a discrete DNA fragment of defined size into the genome precisely in a predefined location or carrying out a discrete deletion of a defined size at a precise location. The technique is based on the observation that DNA single-stranded breaks are preferentially repaired through the HDR pathway, and this reduces the chances of indels (e.g., produced by NHEJ) in the present invention and thus is more efficient than prior art techniques. The invention also provides sequential insertion and/or deletions using single- or double-stranded DNA cutting.

## Claims

### Claim 1

An in vitro method for modifying a genome at a genomic locus of interest in a mouse ES cell, the method comprising:
contacting the mouse ES cell with:
a Cas9 protein;
a CRISPR RNA that hybridizes to a CRISPR target sequence at the genomic locus of interest;
a tracrRNA; and
an incoming nucleic acid sequence that is flanked by:
(i) a 5′ homology arm that is homologous to a 5′ target sequence at the genomic locus of interest; and
(ii) a 3′ homolog arm that is homologous to a 3′ target sequence at the genomic locus of interest;
wherein the incoming nucleic acid sequence is at least 20 kb in size;
wherein following the contacting step, the genome of the mouse ES cell is modified to comprise a targeted genetic modification comprising:
deletion of a region of the genomic locus of interest wherein the deletion is at least 20 kb; and/or
insertion of the insert nucleic acid at the genomic locus of interest wherein the insertion is at least 20 kb.
wherein the targeted genomic modification comprises insertion of:
a. One or more human antibody heavy chain variable domains;
b. One or more human antibody kappa light chain variable domains; or
c. One or more human antibody lambda light chain variable domains;

### Claim 2 (depends on 1)

The method of claim 1, wherein the targeted genomic modification comprises deletion of one or more mouse antibody heavy chain variable domains and insertion of one or more human antibody heavy chain variable domains.

### Claim 3 (depends on 1)

The method of claim 1, wherein the targeted genomic modification comprises deletion of one or more mouse antibody kappa light chain variable domains and insertion of one or more human antibody kappa light chain variable domains.

### Claim 4 (depends on 1)

The method of claim 1, wherein the targeted genomic modification comprises deletion of one or more mouse antibody lambda light chain variable domains and insertion of one or more human antibody lambda light chain variable domains.

### Claim 5 (depends on 1)

The method of claim 1, wherein the mouse ES cell or progeny thereof is developed into a mouse.

### Claim 6 (depends on 1)

A mouse obtained by the method of claim 1.

### Claim 7 (depends on 6)

The mouse of claim 6, wherein the mouse is heterozygous for the targeted genomic modification.

### Claim 8 (depends on 6)

The mouse of claim 6, wherein the mouse is homozygous for the targeted genomic modification.

### Claim 9 (depends on 6)

The mouse of claim 6, wherein the mouse further comprises a targeted modification to insert one or more human antibody kappa light chain variable domains.

### Claim 10 (depends on 6)

The mouse of claim 6, wherein the mouse further comprises a homozygous targeted modification to insert one or more human antibody kappa light chain variable domains.

### Claim 11 (depends on 6)

An antibody produced by the mouse of claim 6.

### Claim 12 (depends on 11)

The antibody according to claim 11, wherein the mouse is heterozygous for the targeted genomic modification.

### Claim 13 (depends on 11)

The antibody according to claim 11, wherein the mouse is homozygous for the targeted genomic modification

### Claim 14 (depends on 11)

The antibody according to claim 11, wherein the mouse further comprises a targeted modification to insert one or more human antibody kappa light chain variable domains.

### Claim 15 (depends on 11)

The antibody according to claim 11, wherein the mouse further comprises a homozygous targeted modification to insert one or more human antibody kappa light chain variable domains.

### Claim 16 (depends on 1)

A method for producing an antibody comprising:
producing a mouse with a modified genome from the ES cell modified by the method of claim 1 or a progeny thereof,
immunizing the mouse with an antigen, and
isolating an antibody produced by the mouse.

### Claim 17 (depends on 16)

The method of claim 16, wherein the targeted genomic modification comprises deletion of one or more mouse antibody heavy chain variable domains and insertion of one or more human antibody heavy chain variable domains

### Claim 18 (depends on 16)

The method of claim 16, wherein the targeted genomic modification comprises deletion of one or more mouse antibody kappa light chain variable domains and insertion of one or more human antibody kappa light chain variable domains.

### Claim 19 (depends on 16)

The method of claim 16, wherein the targeted genomic modification comprises deletion of one or more mouse antibody lambda light chain variable domains and insertion of one or more human antibody lambda light chain variable domains.