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US 9,315,806 B2Granted
Massively parallel combinatorial genetics
AssigneeMassachusetts Institute of Technology
InventorsTimothy Kuan-Ta Lu, Allen Cheng
Life of this patent
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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.
Claims16
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.
3 more elements
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 more elements
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 nth 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.
13 more elements
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 generates compatible ends;
- digesting the vector and insert with restriction enzymes;
- annealing the insert to the vector, thereby producing a combinatorial genetic construct containing multiple DNA elements and multiple barcode elements;
- and
- optionally repeating the method an nth number of times.
10 more elements
Dependencies are inferred from the claim text.