# US 10,501,738 B2 · Automated instrumentation for production of peptide libraries

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,501,738 B2
- Document ID: US10501738B2
- Status: Granted
- Kind: B2
- Application number: 16/393,199
- Filed: 2019-04-24
- Published: 2019-10-24
- Granted: 2019-12-10
- Earliest claimed: 2018-04-24
- Assignees: Inscripta, Inc.
- Applicants: Inscripta, Inc.
- Inventors: Stephen Federowicz; Deanna Church; Michael Graige
- Web page: https://patentgrep.com/patent/US10501738B2

## CPC

- C12N 15/1037: {Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display}
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- C07K 14/723: {G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/815: {for yeasts other than Saccharomyces}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 2310/3517: Marker; Tag
- C40B 40/02: Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
- C40B 50/04: using dynamic combinatorial chemistry techniques
- 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)
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

The present disclosure provides instrumentation and automated methods for creating cell surface display libraries, where the cells of the library display engineered peptides on their cell surfaces for identification of binding pairs. The engineered peptides may be displayed using various cell surface display technologies.

## Claims

### Claim 1

An automated method of creating a cell library expressing engineered peptides using instrumentation, the method comprising:
providing a population of cells;
processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nucleic acid-directed nuclease to create cells comprising nucleic acids that encode engineered peptides configured to be displayed on a surface of the cells;
incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the editing provides nucleic acids that encode engineered peptides in the cells; and
allowing the cells to express and display the engineered peptides on the surface of the cells.

### Claim 2 (depends on 1)

The method of claim 1, wherein the engineered peptides are putative GPCR binding peptides.

### Claim 3 (depends on 1)

The method of claim 1, wherein the engineered peptides are putative ion channel binding peptides.

### Claim 4 (depends on 1)

The method of claim 1, wherein the population of cells are yeast cells.

### Claim 5 (depends on 1)

The method of claim 1, wherein the nuclease is an RNA-directed nuclease.

### Claim 6

An automated method of creating a cell library expressing engineered putative binding peptides on the surface of the cells, the method comprising:
providing a population of cells;
processing the population of cells using an instrument for multiplexed nuclease-directed genome editing using introduced nucleic acids and a nuclease to create cells comprising nucleic acids that encode engineered peptides configured to be displayed on a surface of the cells;
incubating the processed cells to facilitate nucleic acid editing in the cells, wherein the editing provides nucleic acids that encode engineered peptides in the cells; and
allowing the cells to express and display the engineered peptides that are putative target binding peptides on the surface of the cells.

### Claim 7 (depends on 6)

The method of claim 6, wherein the engineered peptides are putative GPCR binding peptides.

### Claim 8 (depends on 6)

The method of claim 6, wherein the engineered peptides comprise putative ion channel binding peptides.

### Claim 9 (depends on 6)

The method of claim 6, wherein the population of cells are yeast cells.

### Claim 10 (depends on 6)

The method of claim 6, wherein the nuclease is a nucleic acid-directed nuclease.

### Claim 11 (depends on 6)

The method of claim 6, wherein the nuclease is an RNA-directed nuclease.

### Claim 12 (depends on 6)

The method of claim 6, wherein one or more of the engineered peptides are binding regions of a target of interest.

### Claim 13 (depends on 6)

The method of claim 6, wherein the engineered peptides are putative ion channel binding peptides.

### Claim 14

An automated, multiplexed method for identifying peptides that selectively bind one or more targets of interest, the method comprising:
providing a population of cells;
processing the population of cells using an automated system for multiplexed nuclease-directed genome editing, wherein the system comprises the steps of:
introducing nucleic acids that encode engineered peptides and a nuclease to a population of cells;
incubating the cells to facilitate nucleic acid editing in the cells; and
allowing the edited cells to express and display the engineered peptides on the surface of the edited cells;
screening the edited cells displaying the engineered peptides against one or more targets of interest; and
identifying the edited cells expressing engineered peptides that selectively bind to one or more targets of interest.

### Claim 15 (depends on 14)

The method of claim 14, further comprising isolating the nucleic acids encoding the engineered peptides that selectively bind to one or more targets of interest from the cells.

### Claim 16 (depends on 14)

The method of claim 14, wherein the nuclease is a nucleic acid-directed nuclease.

### Claim 17 (depends on 14)

The method of claim 14, wherein the nuclease is an RNA-directed nuclease.

### Claim 18 (depends on 14)

The method of claim 14, wherein the cells are identified and/or isolated using a barcode associated with the engineered peptides that selectively bind to one or more targets of interest from the cells.