BIOLOGICAL STATE MACHINES

• US 20170096680A1
• Filed: 09/30/2016
• Published: 04/06/2017
• Est. Priority Date: 10/01/2015
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Roquet, Nathaniel B., Lu, Timothy Kuan-Ta
• Granted Patent: US 10,480,009 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2800/107 for mammalian
• CPC Class Codes: C12N 2800/30 Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

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

1. A system, comprising:

(a) n serine recombinases, wherein n is greater than 2; and

(b) an engineered nucleic acid comprising n-1 pairs of cognate recombination recognition sites (RRSs) for each of the n serine recombinases,

wherein 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.

2. 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.

3. The system of claim 1 [[or 2]], wherein the n serine recombinase is selected from Bxb1, Tp901, A 118, Ph1F and AraC.

4. 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.

5. 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.

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

7. The system of claim 6, wherein the at least one promoter is selected from P_PhlF, P_BAD and P_Lteto.

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

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

12. A cell comprising the system of claim 1.

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

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

15. A method of using the system of claim 1 as a therapeutic device or as a diagnostic device.

16. A method of using the system of claim 1 to control differentiation of a cell.

17. A method of using the system of claim 1 to detect chemical signals in a cell.

10. A system, comprising:

(a) three serine recombinases; and

(b) an engineered nucleic acid comprising two pairs of cognate recombinase recognition sites (RRSs) for each of the three serine recombinase,

wherein 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.

11. A system, comprising:

(a) four serine recombinases; and

(b) an engineered nucleic acid comprising three pairs of cognate recombinase recognition sites (RRSs) for each of the four serine recombinase,

wherein 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.