# US 10,480,009 B2 · Biological state machines

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- Number: US 10,480,009 B2
- Document ID: US10480009B2
- Status: Granted
- Kind: B2
- Application number: 15/283,162
- Filed: 2016-09-30
- Published: 2017-04-06
- Granted: 2019-11-19
- Earliest claimed: 2015-10-01
- Assignees: Massachusetts Institute of Technology
- Applicants: Massachusetts Institute of Technology
- Inventors: Timothy Kuan-Ta Lu; Nathaniel B. Roquet
- Web page: https://patentgrep.com/patent/US10480009B2

## CPC

- C12N 15/85: for animal cells
- C12N 9/16: acting on ester bonds (3.1)
- G16B 30/00: ICT specially adapted for sequence analysis involving nucleotides or amino acids
- C12N 2800/107: for mammalian
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G16B: BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY

## Abstract

Provided herein are recombinase-based frameworks for building state machines in vitro and in vivo by using chemically controlled DNA excision and inversion operations to encode state in DNA sequence.

## Claims

### Claim 1

A system, comprising:
(a) n serine recombinases, wherein n is greater than 2; and
(b) an engineered nucleic acid comprising n(n−1) pairs of cognate recombination recognition sites (RRS),
wherein the n(n−1) pairs of RRSs of (b) are arranged in an overlapping configuration such that the two RRSs of each pair of the n(n−1) pairs of RRSs are separated from each other by at least one RRS of another pair of the n(n−1) pairs of RRSs, and wherein recombination between the two RRSs of each pair of the n(n−1) pairs of RRSs either inverts or excises at least one RRS of another pair of the n(n−1) pairs of RRSs.

### Claim 2 (depends on 1)

The system of claim 1, wherein n is greater than or equals 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

### Claim 3 (depends on 1)

The system of claim 1, wherein the n serine recombinase is selected from Bxb1, Tp901, A118, PhlF and AraC.

### Claim 4 (depends on 1)

The system of claim 1, wherein the RRSs are selected from an attB site, an attP site, an attB site modified to include a CA dinucleotide, an attP site modified to include a CA dinucleotide, an attB site modified to include a GT dinucleotide, an attP site modified to include a GT dinucleotide, an attB site modified to include a AG dinucleotide, an attP site modified to include a AG dinucleotide, an attB site modified to include a TC dinucleotide, an attP site modified to include a TC dinucleotide, an attB site modified to include a AA dinucleotide, an attP site modified to include a AA dinucleotide, an attB site modified to include a GG dinucleotide, and an attP site modified to include a GG dinucleotide.

### Claim 5 (depends on 1)

The system of claim 1, wherein the system further comprises at least one engineered nucleic acid comprising at least one promoter operably linked to a nucleotide sequence encoding at least one of the n serine recombinases.

### Claim 6 (depends on 5)

The system of claim 5, wherein the at least one promoter is inducible.

### Claim 7 (depends on 6)

The system of claim 6, wherein the at least one promoter is selected from PPhlF, PBAD and PLtetO.

### Claim 8 (depends on 1)

The system of claim 1, wherein the engineered nucleic acid of (b) further comprises a nucleotide sequence encoding a detectable molecule.

### Claim 9 (depends on 8)

The system of claim 8, wherein the detectable molecule is a fluorescent molecule.

### Claim 10

A system, comprising:
(a) three serine recombinases; and
(b) an engineered nucleic acid comprising six pairs of cognate recombinase recognition sites (RRSs),
wherein the six pairs of RRSs of (b) are arranged in an overlapping configuration such that the two RRSs of each pair of the six pairs of RRSs are separated from each other by at least one RRS of another pair of the six pairs of RRSs, and wherein recombination between the two RRSs of each pair of the six pairs of RRSs either inverts or excises at least one RRS of another pair of the six pairs of RRSs.

### Claim 11

A system, comprising:
(a) four serine recombinases; and
(b) an engineered nucleic acid comprising twelve pairs of cognate recombinase recognition sites (RRSs),
wherein the twelve pairs of RRSs of (b) are arranged in an overlapping configuration such that the two RRSs of each pair of the twelve pairs of RRSs are separated from each other by at least one RRS of another pair of the twelve pairs of RRSs, and wherein recombination between the two RRSs of each pair of the twelve pairs of RRSs either inverts or excises at least one RRS of another pair of the twelve pairs of RRSs.

### Claim 12 (depends on 1)

An isolated cell comprising the system of claim 1.

### Claim 13 (depends on 12)

The isolated cell of claim 12, wherein the isolated cell is a bacterial cell or a mammalian cell.

### Claim 14 (depends on 12)

The isolated cell of claim 12, wherein the isolated cell is a stem cell.