Back to results
US 2019/0100769 A1Published application
Massively parallel combinatorial genetics for crispr
AssigneeMassachusetts Institute of Technology
InventorsTimothy Kuan-Ta Lu, Alan Siu Lun Wong, Ching Gee Choi
Life of this patent
Estimating the term…
Abstract
Described herein are methods and compositions that enable rapid generation of high-order combinations of genetic elements comprising a CRISPR guide sequence and a scaffold sequence, and a barcode for rapid identification of the combination of genetic elements encoded within a single cell or a pooled population. Also described herein compositions of inhibitors of epigenetic genes and methods for reducing cell proliferation and/or treating cancer.
Claims52
A genetic construct comprising
- a first DNA element comprising
- a CRISPR guide sequence and
- a scaffold sequence;
- a first compatible end element and a second compatible end element flanking the first 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, the third compatible end element, and the fourth compatible end element are on the other side of the separation site.
5 more elements
A genetic construct comprising:
- a plurality of DNA elements, wherein each DNA element of the plurality of DNA element comprises a CRISPR guide sequence and a scaffold sequence;
- 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 vector, comprising:
- (a) providing a vector containing a first genetic construct comprising:
- a CRISPR guide sequence;
- a second compatible end element and a first recognition site for a first restriction enzyme flanking the CRISPR guide sequence;
- a barcode element;
- and
- a third compatible end element and a second recognition site for a second restriction enzyme flanking the barcode element;
- (b) cleaving the first genetic construct at the first recognition site, resulting in a fifth compatible end element, and cleaving the vector at the second recognition site, resulting in a sixth compatible end element;
- (c) providing a scaffold element comprising
- a scaffold sequence;
- a separation site comprising a first compatible end element and a fourth compatible end element;
- and
- a seventh compatible end element and an eighth compatible end element flanking the scaffold element, wherein the seventh compatible end element is capable of annealing to the fifth compatible end element and the eighth compatible end element is capable of annealing to the sixth compatible end element;
- (d) annealing the scaffold element to the cleaved first genetic construct, wherein the annealing occurs at compatible end elements within the vector and the scaffold element that are capable of annealing to each other, and wherein after the annealing, the scaffold element is integrated between the CRISPR guide sequence and the barcode element, and wherein the separation site is located between the scaffold sequence and the barcode element, creating a combinatorial vector.
10 more elements
A genetic construct comprising
- at least two CRISPR guide sequences;
- a barcode element;
- and
- a restriction recognition site located between each CRISPR guide sequence and between the barcode element and the CRISPR guide sequence nearest to the barcode element.
1 more element
A genetic construct comprising
- a plurality of DNA elements, each comprising
- a CRISPR guide sequence and
- a scaffold sequence;
- a barcode element;
- and
- a promoter sequence located upstream of each of the DNA elements of the plurality of DNA elements.
3 more elements
A method for generating a combinatorial vector, comprising
- (a) providing a vector comprising:
- a plurality of CRISPR guide sequences;
- a barcode element, wherein the barcode element is located downstream of the plurality of CRISPR guide sequences;
- optionally a promoter sequence located upstream of at least one of the plurality of CRISPR guide sequences;
- and
- a plurality of recognition sites for a plurality of restriction enzymes, wherein each of the plurality of recognition sites is located downstream of one of the plurality of CRISPR guide sequences;
- (b) cleaving the vector at at least one of the plurality of recognition sites with at least one of the plurality of restriction enzymes, resulting in a first compatible end element and a second compatible end element;
- (c) providing a first scaffold element comprising:
- a scaffold sequence,
- optionally a promoter sequence, and
- a third compatible end element and fourth compatible end element flanking the first scaffold element, wherein the third compatible end element is capable of annealing to the first compatible end element of the cleaved vector and the fourth compatible end element is capable of annealing to the second compatible end element of the cleaved vector;
- (d) annealing the first scaffold element to the cleaved vector, wherein the annealing occurs at compatible end elements within the first scaffold element and the cleaved vector, and wherein after annealing, the first scaffold element is integrated downstream of one of the plurality of CRISPR guide sequences, thereby producing a combinatorial vector.
9 more elements
A method for generating a combinatorial vector, comprising
- (a) providing a vector comprising:
- a plurality of CRISPR guide sequences,
- a barcode element, wherein the barcode element is located upstream of the plurality of CRISPR guide sequences;
- optionally a promoter sequence located upstream of at least one of the plurality of CRISPR guide sequences;
- and
- a plurality of recognition sites for a plurality of restriction enzymes, wherein each of the plurality of recognition sites is located upstream of one of the plurality of CRISPR guide sequences;
- (b) cleaving the vector at least one of the plurality of recognition sites with at least one of the plurality of restriction enzymes, resulting in a first compatible end element and a second compatible end element;
- (c) providing a first scaffold element comprising:
- optionally a scaffold sequence,
- a promoter sequence, and
- a third compatible end element and fourth compatible end element flanking the first scaffold element, wherein the third compatible end element is capable of annealing to the first compatible end element of the cleaved vector and the fourth compatible end element is capable of annealing to the second compatible end element of the cleaved vector;
- (d) annealing the first scaffold element to the cleaved vector, wherein the annealing occurs at compatible end elements within the first scaffold element and the cleaved vector, and wherein after annealing, the first scaffold element is integrated upstream of one of the plurality of CRISPR guide sequences, thereby producing a combinatorial vector.
9 more elements
A composition comprising two or more inhibitors targeting two or more epigenetic genes selected from the combinations of epigenetic genes set forth in Table 2.
A method for reducing proliferation of a cell, comprising contacting the cell with a combination of two or more inhibitors targeting two or more epigenetic genes selected from the combinations of epigenetic genes set forth in Table 2.
A method for treating cancer in a subject, comprising administering to the subject a combination of two or more inhibitors targeting two or more epigenetic genes selected from the combinations of epigenetic genes set forth in Table 2, wherein each of the two or more inhibitors are administered in an effective amount.
A method for identifying a combination of inhibitors of epigenetic genes that reduces proliferation of a cell, the method comprising:
- contacting a first population of cells and a second population of cells with a plurality of combinations of two or more CRISPR guide sequences and scaffold sequences and a Cas9 endonuclease;
- culturing the first population of cells and the second population of cells such that the second population of cells is cultured for a longer duration compared to the first population of cells;
- identifying the combinations of two or more CRISPR guide sequences and scaffold sequences in the first population of cells and the combinations of two or more CRISPR guide sequences and scaffold sequences in the second population of cells;
- comparing the abundance of each combination of two or more CRISPR guide sequences and scaffold sequences in the first population of cells to the abundance of each combination of two or more CRISPR guide sequences and scaffold sequences in the second population of cells;
- and
- identifying a combination of two or more CRISPR guide sequences and scaffold sequences that is absent from or in reduced abundance in the second population of cells but present in or in increased abundance in the first population of cells as a combination of CRISPR guide sequences and scaffold sequences that reduces cell proliferation.
3 more elements
A method for identifying a combination of genes to be inhibited to reduce proliferation of a cell, the method comprising:
- contacting a first population of cells and a second population of cells with a plurality of combinations of two or more CRISPR guide sequences and scaffold sequences and a Cas9 endonuclease;
- culturing the first population of cells and the second population of cells such that the second population of cells is cultured for a longer duration compared to the first population of cells;
- identifying the combinations of two or more CRISPR guide sequences and scaffold sequences in the first population of cells and the combinations of two or more CRISPR guide sequences and scaffold sequences in the second population of cells;
- comparing the abundance of each combination of two or more CRISPR guide sequences and scaffold sequences in the first population of cells to the abundance of each combination of two or more CRISPR guide sequences and scaffold sequences in the second population of cells;
- and
- identifying a combination of two or more CRISPR guide sequences and scaffold sequences that is absent from or in reduced abundance in the second population of cells but present in or in increased abundance in the first population of cells as a combination of genes to be inhibited to reduce proliferation.
3 more elements
Dependencies are inferred from the claim text.