Massively parallel combinatorial genetics

Patent metadata

Field	Value
Publication number	US-9315806-B2
Application number	US-201314411628-A
Country	US
Kind code	B2
Filing date	Jun 28, 2013
Priority date	Jun 29, 2012
Publication date	Apr 19, 2016
Grant date	Apr 19, 2016

Abstract

The invention relates to methods and compositions that enable the rapid generation of high-order combinations of genetic elements, and that provide a barcoded basis for rapid characterization of the specific combination of genetic elements encoded within a single cell or in a pooled population.

First claim

What is claimed is:

1. A genetic construct comprising:
a DNA element;
a first compatible end element and a second compatible end element flanking the DNA element, wherein the first and second compatible end elements are capable of annealing to each other;
a barcode element;
a third compatible end element and a fourth compatible end element flanking the barcode element, wherein the third and fourth compatible end elements are capable of annealing to each other but are not capable of annealing to the first or second compatible end elements; and
a separation site located between the fourth compatible end element and the first compatible end element, wherein the DNA element, first compatible end element and second compatible end element are on one side of the separation site, and the barcode element, third compatible end element and fourth compatible end element are on the other side of the separation site.

2. A genetic construct comprising:
a plurality of DNA elements;
a first compatible end element and a second compatible end element flanking the plurality of DNA elements, wherein the first and second compatible end elements are capable of annealing to each other;
a plurality of barcode elements;
a third compatible end element and a fourth compatible end element flanking the plurality of barcode elements, wherein the third and fourth compatible end elements are capable of annealing to each other but are not capable of annealing to the first or second compatible end elements; and
a separation site located between the plurality of DNA elements and the plurality of barcode elements.

3. A method for generating a combinatorial genetic construct, comprising:
providing a vector containing a first genetic construct according to claim 1 ;
cleaving the vector at the separation site within the first genetic construct, resulting in the first genetic construct being separated into first and second segments;
providing a second genetic construct according to claim 1 ; and
annealing the second genetic construct to the cleaved vector, wherein the annealing occurs at compatible end elements within the first and second genetic constructs that are capable of annealing to each other, and wherein after annealing, the second genetic construct is integrated between the first and second segments of the first genetic construct, creating a combinatorial genetic construct.

4. The method of claim 3 , wherein the method is iterative.

5. A method for identification of a DNA element or a plurality of DNA elements, comprising:
providing a genetic construct according to claim 1 ;
conducting an assay to determine the DNA sequence of the barcode or plurality of barcodes within the genetic construct and/or the DNA sequence of the DNA element or plurality of DNA elements within the genetic construct; and
identifying the DNA element or plurality of DNA elements.

6. A library comprising:
two or more genetic constructs according to claim 1 .

7. A method for generating a combinatorial genetic construct, comprising:
providing a vector comprising:
a first DNA element,
a first barcode element, and
two site-specific recombination elements located between the first DNA element and the first barcode element;

providing a first insert comprising:
a second DNA element,
a second barcode element, and
site-specific recombination elements flanking each of the second DNA element and the second barcode element, such that two site-specific recombination elements are located between the second DNA element and the second barcode element that are not compatible with the site-specific recombination elements within the vector, and two site-specific recombination elements are located outside of the second DNA element and the second barcode element that are compatible with the site-specific recombination elements within the vector;

conducting site specific recombination between the vector and the first insert, wherein the site specific recombination occurs between the site-specific recombination elements within the vector located between the first DNA element and the first barcode element and the compatible site-specific recombination elements within the first insert located outside of the second DNA element and the second barcode element, and wherein following site-specific recombination, the first insert is located within the vector, and the vector contains multiple DNA elements and multiple barcode elements, with two site-specific recombination elements located between the multiple DNA elements and the multiple barcode elements;
providing a second insert comprising:
a third DNA element,
a third barcode element, and
site-specific recombination elements flanking each of the third DNA element and the third barcode element, such that two site-specific recombination elements are located between the third DNA element and the third barcode element that are not compatible with the two site-specific recombination elements located between the multiple DNA elements and the multiple barcode elements of the vector, and two site-specific recombination elements are located outside of the third DNA element and the third barcode element that are compatible with the two site-specific recombination elements located between the multiple DNA elements and the multiple barcode elements of the vector;

conducting site specific recombination between the vector and the second insert, wherein the site specific recombination occurs between the site-specific recombination elements within the vector located between the multiple DNA elements and the multiple barcode elements and the compatible site-specific recombination elements within the second insert located outside of the third DNA element and the third barcode element, and wherein following site-specific recombination, the second insert is located within the vector, and the vector contains multiple DNA elements and multiple barcode elements, with two site-specific recombination elements located between the multiple DNA elements and the multiple barcode elements; and
repeating the site-specific recombination an n th number of times, alternating between site-specific recombination between the vector and the first insert and site-specific recombination between the vector and the second insert, thereby creating a combinatorial genetic construct.

8. A combinatorial genetic construct produced by the method of claim 7 .

9. A method for identification of a DNA element or a plurality of DNA elements, comprising:
providing a combinatorial genetic construct according to claim 8 ;
conducting an assay to determine the DNA sequence of one or more barcode elements within the combinatorial genetic construct and/or the DNA sequence of one or more DNA elements within the combinatorial genetic construct; and
identifying the DNA element or plurality of DNA elements.

10. A method for generating a combinatorial genetic construct, comprising:
providing a vector comprising:
a first DNA element,
a first barcode element, and
a recognition site for a first restriction enzyme located between the first DNA element and the first barcode element;

providing an insert comprising:
a second DNA element,
a second barcode element,
a recognition site for the first restriction enzyme located between the second DNA element and the second barcode element, and
two recognition sites for one or more restriction enzymes that are distinct from the first restriction enzyme located outside of the second DNA element and second barcode element, such that restriction digestion at the recognition site within the vector and at the two recognition sites located outside of the second DNA element and second barcode element within the insert gener

Assignees

• Massachusetts Inst Technology

Inventors

• Lu Timothy Kuan-Ta
• Cheng Allen

Classifications

• C12N15/1065 Primary

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q2525/131

  incorporating a restriction site · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• G01N2035/00752

  bar codes · CPC title

• C12N15/1079

  Screening libraries by altering the phenotype or phenotypic trait of the host (reporter assays C12N15/1086) · CPC title