# US 11,999,767 B2 · Target recognition motifs and uses thereof

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- Number: US 11,999,767 B2
- Document ID: US11999767B2
- Status: Granted
- Kind: B2
- Application number: 17/133,141
- Filed: 2020-12-23
- Published: 2021-04-22
- Granted: 2024-06-04
- Earliest claimed: 2018-04-03
- Assignees: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Applicants: The Broad Institute, Inc.; Massachusetts Institute of Technology
- Inventors: Feng Zhang
- Web page: https://patentgrep.com/patent/US11999767B2

## CPC

- C07K 14/24: from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- 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 15/1086: {Preparation or screening of expression libraries, e.g. reporter assays}
- C12N 15/86: Viral vectors
- C07K 2319/01: containing a localisation/targetting motif
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C07K 2319/70: containing domain for protein-protein interaction
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The disclosure provides novel programmable targeting sequences and applications thereof. The targeting sequences can be engineered for binding to proteins, polypeptides, and other macromolecules.

## Claims

### Claim 1

A method of engineering a protein or polypeptide to bind to a target of interest, which comprises:
inserting one or more engineered, heterologous Photorhabdus tandem repeat protein target recognition sequence (TRS) motif(s) in the protein or polypeptide, and modifying the one or more engineered, heterologous Photorhabdus TRS motif(s);
associating the one or more modified, engineered, heterologous Photorhabdus TRS motif(s) with a detectable marker; and
detecting whether the engineered protein or polypeptide binds to the target; or
detecting whether the engineered protein or polypeptide exhibits enzymatic activity against the target.

### Claim 2 (depends on 1)

The method of claim 1, wherein the one or more modified, engineered, heterologous Photorhabdus TRS motif(s) comprises:
X1(E/K)(N/D)X2(S/T)X3 (SEQ ID NO: 1) flanked by a hydrophobic region and a charged region;
wherein X1 comprises 1 to 7 amino acid(s) in length and comprises any amino acid(s),
wherein X2 comprises 0 to 12 amino acid(s) in length and comprises any amino acid(s), and
wherein X3 comprises 1 to 7 amino acid(s) in length and comprises any amino acid(s),
wherein the hydrophobic region comprises at least one hydrophobic amino acid residue, and
wherein the charged region comprises at least one charged amino acid residue.

### Claim 3 (depends on 2)

The method of claim 2, wherein the hydrophobic region is at the N-terminus and comprises G(V/A/L)G and the charged region is at the C-terminus and comprises K(K/E)Q.

### Claim 4 (depends on 1)

The method of claim 1, wherein the protein or polypeptide comprises a functional domain.

### Claim 5 (depends on 1)

The method of claim 1, wherein the protein comprises an insecticidal toxin or a component thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the one or more modified, engineered, heterologous Photorhabdus TRS motif(s) and the detectable marker are covalently linked.

### Claim 7 (depends on 1)

The method of claim 1, wherein the target is a polypeptide or polynucleotide.