Multiplex RNA-guided genome engineering
First Claim
1. A method of making multiple alterations to target DNA in a eukaryotic cell constitutively expressing a Cas9 enzyme that forms a co-localization complex with a guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner comprising(a) introducing into the cell consitutively expressing the Cas9 enzyme a plurality of guide RNAs complementary to different sites of the target DNA, wherein each of the plurality of guide RNAs is a tracrRNA-crRNA fusion, wherein each of the plurality of guide RNAs and the Cas9 enzyme are members of co-localization complexes for the target DNA, wherein the plurality of guide RNAs is introduced without also introducing a foreign nucleic acid encoding the Cas9 enzyme,introducing into the cell constitutively expressing the Cas9 enzyme a plurality of donor nucleic acid sequences,wherein at least one of the plurality of guide RNAs and the Cas9 enzyme co-localize to a site of the target DNA, the Cas9 enzyme cleaves the target DNA and one of the plurality of donor nucleic acid sequences is inserted into the target DNA at the site of cleavage to produce altered DNA in the cell, and(b) repeating step (a) to produce multiple alterations to the DNA in the cell.
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Abstract
Methods of multiplex genome engineering in cells using Cas9 is provided which includes a cycle of steps of introducing into the cell a first foreign nucleic acid encoding one or more RNAs complementary to the target DNA and which guide the enzyme to the target DNA, wherein the one or more RNAs and the enzyme are members of a co-localization complex for the target DNA, and introducing into the cell a second foreign nucleic acid encoding one or more donor nucleic acid sequences, and wherein the cycle is repeated a desired number of times to multiplex DNA engineering in cells.
6 Citations
9 Claims
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1. A method of making multiple alterations to target DNA in a eukaryotic cell constitutively expressing a Cas9 enzyme that forms a co-localization complex with a guide RNA complementary to the target DNA and that cleaves the target DNA in a site specific manner comprising
(a) introducing into the cell consitutively expressing the Cas9 enzyme a plurality of guide RNAs complementary to different sites of the target DNA, wherein each of the plurality of guide RNAs is a tracrRNA-crRNA fusion, wherein each of the plurality of guide RNAs and the Cas9 enzyme are members of co-localization complexes for the target DNA, wherein the plurality of guide RNAs is introduced without also introducing a foreign nucleic acid encoding the Cas9 enzyme, introducing into the cell constitutively expressing the Cas9 enzyme a plurality of donor nucleic acid sequences, wherein at least one of the plurality of guide RNAs and the Cas9 enzyme co-localize to a site of the target DNA, the Cas9 enzyme cleaves the target DNA and one of the plurality of donor nucleic acid sequences is inserted into the target DNA at the site of cleavage to produce altered DNA in the cell, and (b) repeating step (a) to produce multiple alterations to the DNA in the cell.
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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InventorsChurch, George M., Dicarlo, James
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Primary Examiner(s)Hibbert, Catherine S
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Application NumberUS14/903,719Publication NumberTime in Patent Office3,010 DaysField of SearchUS Class CurrentCPC Class CodesC12N 15/111 General methods applicable ...C12N 15/902 using homologous recombinationC12N 2310/20 involving clustered regular...
