Large Gene Excision and Insertion

• US 20150140664A1
• Filed: 06/30/2014
• Published: 05/21/2015
• Est. Priority Date: 11/19/2013
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: CHURCH, George M., BYRNE, Susan M.
• Granted Patent: US 10,787,684 B2
• Time in Patent Office: Days
• US Class Current: 435/462
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

Methods of simultaneously excising large nucleic acid sequences from a target nucleic acid and inserting large foreign nucleic sequences into the target nucleic acid sequence using DNA binding protein nucleases are described.

Claims

1. A method of altering a target nucleic acid in a cell comprising

introducing into the cell a first foreign nucleic acid encoding one or more guide RNA sequences complementary to DNA, wherein the DNA includes the target nucleic acid,

introducing into the cell a second foreign nucleic acid encoding a Cas9 protein that binds to the DNA and is guided by the one or more guide RNA sequences,

introducing into the cell a third foreign nucleic acid encoding an exogenous nucleic acid sequence to be included into the target nucleic acid sequence,

wherein the one or more guide RNA sequences, the Cas9 protein, and the exogenous nucleic acid sequence are expressed,

wherein the one or more guide RNA sequences and the Cas9 protein co-localize to the DNA and wherein the Cas9 protein creates two or more double stranded breaks to remove a first nucleic acid sequence of interest and wherein the exogenous nucleic acid sequence is inserted between the two break points of the target nucleic acid.

2. The method of claim 1 wherein the exogenous nucleic acid sequence to be included into the target nucleic acid sequence is flanked by sequences complementary to the area around the gene replacement.

3. The method of claim 1 wherein the exogenous nucleic acid is between greater than 100 base pairs and about 100,000 base pairs in length.

4. The method of claim 1 wherein the first nucleic acid sequence of interest is between greater than 100 base pairs and about 10,000 base pairs in length.

5. The method of claim 1 wherein the cell is a eukaryotic cell.

6. The method of claim 1 wherein the cell is a yeast cell, a plant cell or an animal cell.

7. The method of claim 1 wherein the guide RNA is between about 10 to about 500 nucleotides.

8. The method of claim 1 wherein the guide RNA is between about 20 to about 100 nucleotides.

9. The method of claim 1 wherein the one or more guide RNA sequences is a crRNA.

10. The method of claim 1 wherein the one or more guide RNA sequences is a tracrRNA-crRNA fusion.

11. The method of claim 1 wherein the DNA is genomic DNA, mitochondrial DNA, viral DNA, or exogenous DNA.

12. The method of claim 1 wherein the exogenous nucleic acid sequence is inserted into the target nucleic acid sequence by homologous recombination.

13. The method of claim 1 wherein the exogenous nucleic acid sequence is inserted into the target nucleic acid sequence by nonhomologous end joining.