Multiplexed genetic reporter assays and compositions

Patent metadata

Field	Value
Publication number	US-11767534-B2
Application number	US-201214115608-A
Country	US
Kind code	B2
Filing date	May 4, 2012
Priority date	May 4, 2011
Publication date	Sep 26, 2023
Grant date	Sep 26, 2023

Abstract

The invention provides methods for determining the activity of a plurality of nucleic acid regulatory elements. These methods may facilitate, e.g., the systematic reverse engineering, and optimization of mammalian cis-regulatory elements at high resolution and at a large scale. The method may include integration of multiplexed DNA synthesis and sequencing technologies to generate and quantify the transcriptional regulatory activity of e.g., thousands of arbitrary DNA sequences in parallel in cell-based as says (e.g., mammalian cell based assays).

First claim

What is claimed is:

1. A plurality of expression vectors,
wherein each of said expression vectors comprises a nucleic acid regulatory element, an open reading frame, a pair of universal primer sites, and one or more identifying nucleic acid tags;
wherein the nucleic acid regulatory element, the open reading frame, and the one or more identifying nucleic acid tags are between the pair of universal primer sites;
wherein the open reading frame is located 3′ to the nucleic acid regulatory element and the one or more identifying nucleic acid tags are located 3′ to the open reading frame;
wherein the open reading frame of each of said plurality of expression vectors is identical, and optionally encodes a fluorescent protein or a luciferase;
wherein the plurality of expression vectors comprises a plurality of distinct nucleic acid regulatory elements, wherein the plurality of distinct regulatory elements comprises synthetic variants of a single regulatory element;
wherein each nucleic acid regulatory element is paired with the one or more identifying nucleic acid tags; and
wherein the pair of universal primer sites comprise a first primer site comprising the sequence of SEQ ID NO: 5 and a second primer site comprising the sequence of SEQ ID NO: 7.

2. A population of cells comprising the plurality of expression vectors of claim 1 .

3. The plurality of expression vectors of claim 1 , wherein each identifying tag comprises a sequence that is unique over a stretch of the nucleic acid tags, wherein the sequence that is unique may be continuous along the length of the nucleic acid tag sequence or the nucleic acid tag may include stretches of nucleic acid sequence that are not unique to any one tag.

4. The plurality of expression vectors of claim 1 , wherein each expression vector further comprises an identical stretch of nucleotides located 3′ to the identifying nucleic acid tags, and wherein optionally said identical stretch of nucleotides comprises a transcriptional terminator or poly-adenylation signal.

5. The plurality of expression vectors of claim 1 , wherein (i) the variants of a single regulatory element differ from one another by a single nucleotide substitution, deletion, or insertion, optionally wherein among said variants of a single regulatory element are variant regulatory elements comprising single nucleotide substitutions of every nucleotide of said single regulatory element, or (ii) wherein each variant of a single regulatory element differs from the remaining variants of the single regulatory element by two or more single nucleotide substitutions, deletions, insertions, or combinations thereof.

6. The plurality of expression vectors of claim 1 , wherein said plurality of expression vectors comprises at least 100 or more distinct expression vectors, and wherein the identifying nucleic acid tags are oriented in each of the plurality of expression vectors such that the identifying nucleic acid tags are transcribed in the same mRNA transcript as the associated open reading frame.

7. The plurality of expression vectors of claim 6 , wherein said plurality of expression vectors comprises at least 500 distinct expression vectors, wherein the at least 500 distinct expression vectors each comprises a distinct nucleic acid regulatory element comprising a synthetic variant of a single regulatory element.

8. The plurality of expression vectors of claim 1 , wherein each expression vector further comprises regions that facilitate stable integration into a cellular genome.

9. A method of determining individual activities of a plurality of nucleic acid regulatory elements, the method comprising:
introducing the plurality of expression vectors of claim 1 into cells in which said open reading frames and said tags are expressed, or providing cells with said plurality of expression vectors so introduced; and
determining expression of said tags expressed in the cells; wherein the amount of each tag detected is an indication of the activity of a corresponding nucleic acid regulatory element.

10. The method of claim 9 , further comprising isolating mRNA from said cells prior to said determining the amount of said tags expressed in said cells.

11. The method of claim 10 , wherein said mRNA isolated by poly-A isolation.

12. The method of claim 11 , further comprising first strand cDNA synthesis using said isolated mRNA as a template.

13. The method of claim 12 , wherein said determining the amount of said tags expressed in the cells comprises quantitatively sequencing the nucleic acid molecules resulting from said cDNA synthesis.

14. The method of claim 13 , further comprising determining the amount of each tag in said plurality of expression vectors by quantitatively sequencing said plurality of expression vectors.

15. The method of claim 14 , further comprising normalizing the amount of said tags expressed in the cells against the amount of each of said tags in said plurality of expression vectors.

16. The method of claim 10 , wherein said determining the amount of said tags expressed in the cells comprises determining the quantity of mRNA hybridized to nucleic acid molecules complementary to said tags.

17. The method of claim 9 , wherein each distinct regulatory element differs from the remaining distinct regulatory elements by a single nucleotide substitution, deletion, or insertion;
and wherein among said distinct regulatory elements are regulatory elements comprising single nucleotide substitutions of every nucleotide of said single regulatory element; or wherein said distinct regulatory elements comprisE regulatory elements that differ from said single regulatory element by one or more transversions or transpositions of stretches of nucleic acid sequences of greater than 4 nucleotides.

18. A plurality of nucleic acid constructs comprised within expression vectors,
wherein the plurality of nucleic acid constructs comprises a plurality of distinct nucleic acid regulatory elements, wherein the distinct regulatory elements comprise synthetic variants of a single regulatory element;
wherein each of said constructs comprises one or more identifying nucleic acid tags, two or more restriction enzyme sites, a pair of universal primer sites, and a particular nucleic acid regulatory element paired to said one or more nucleic acid tags,
wherein the nucleic acid regulatory element, two or more restriction enzyme sites, and one or more identifying nucleic acid tags are between the pair of universal primer sites;
wherein the two or more restriction enzyme sites are located 3′ to the nucleic acid regulatory element and the one or more identifying nucleic acid tags are located 3′ to the two or more restriction enzyme sites; and
wherein the pair of universal primer sites comprise a first primer site comprising the sequence of SEQ ID NO: 5 and a second primer site comprising the sequence of SEQ ID NO: 7.

19. A method of determining individual activities of a plurality of nucleic acid regulatory elements, the method comprising:
providing the plurality of expression vectors of claim 18 ;
introducing said expression vectors into cells in which said open reading frames and said tags are expressed; and
determining the amount of said tags expressed in the cells; wherein the amount of each tag detected is an indication of the activity of a corresponding nucleic acid regulatory element.

20. The method of claim 19 , further comprising
identifying variants of a nucleic acid regulatory element that have higher individual activities or higher rel

Assignees

• Mikkelsen Tarjei
• Gnirke Andreas
• Melnikov Alexandre
• Lander Eric S
• Wang Li
• Zhang Xiaolan
• Broad Inst Inc

Inventors

• Mikkelsen Tarjei
• Gnirke Andreas
• Melnikov Alexandre
• Lander Eric S
• Wang Li
• Zhang Xiaolan

Classifications

• C12N15/63 Primary

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• C12N15/1086

  Preparation or screening of expression libraries, e.g. reporter assays · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title