Large gene excision and insertion
First Claim
1. A method of altering a target nucleic acid in a human cell comprisingintroducing into the human cell one or more first foreign nucleic acids encoding two or more guide RNA sequences complementary to DNA at positions defining a nucleic acid sequence of greater than 1000 base pairs, wherein the nucleic acid sequence includes the target nucleic acid,introducing into the human cell a second foreign nucleic acid encoding a Cas9 protein that binds to the DNA and is guided by the two or more guide RNA sequences,introducing into the human cell an exogenous nucleic acid sequence of between greater than 1000 base pairs and about 100,000 base pairs in length to be included into the target nucleic acid sequence, wherein the exogenous nucleic acid sequence is flanked by homology arm sequences between about 1500 to about 2500 bases in length complementary to either side of the target nucleic acid to be removed,wherein the two or more guide RNA sequences and the Cas9 protein are expressed,wherein the two 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 the nucleic acid sequence of greater than 1000 base pairs and wherein the exogenous nucleic acid sequence is inserted between the two break points of the DNA by homologous recombination.
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Accused Products
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.
33 Citations
34 Claims
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1. A method of altering a target nucleic acid in a human cell comprising
introducing into the human cell one or more first foreign nucleic acids encoding two or more guide RNA sequences complementary to DNA at positions defining a nucleic acid sequence of greater than 1000 base pairs, wherein the nucleic acid sequence includes the target nucleic acid, introducing into the human cell a second foreign nucleic acid encoding a Cas9 protein that binds to the DNA and is guided by the two or more guide RNA sequences, introducing into the human cell an exogenous nucleic acid sequence of between greater than 1000 base pairs and about 100,000 base pairs in length to be included into the target nucleic acid sequence, wherein the exogenous nucleic acid sequence is flanked by homology arm sequences between about 1500 to about 2500 bases in length complementary to either side of the target nucleic acid to be removed, wherein the two or more guide RNA sequences and the Cas9 protein are expressed, wherein the two 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 the nucleic acid sequence of greater than 1000 base pairs and wherein the exogenous nucleic acid sequence is inserted between the two break points of the DNA by homologous recombination.
Specification
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- Resources
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Current AssigneePresident and Fellows of Harvard College (Harvard Corporation)
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Original AssigneePresident and Fellows of Harvard College (Harvard Corporation)
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InventorsByrne, Susan M., Church, George M.
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Primary Examiner(s)Wu, Julie
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Application NumberUS14/319,693Publication NumberTime in Patent Office2,283 DaysField of SearchNoneUS Class CurrentCPC Class CodesC12N 15/102 Mutagenizing nucleic acidsC12N 15/907 in mammalian cells
