RNA-programmed genome editing in human cells
Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide...
| Main Authors: | , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
eLife Sciences Publications Ltd
2013-01-01
|
| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/00471 |
| _version_ | 1811236853203861504 |
|---|---|
| author | Martin Jinek Alexandra East Aaron Cheng Steven Lin Enbo Ma Jennifer Doudna |
| author_facet | Martin Jinek Alexandra East Aaron Cheng Steven Lin Enbo Ma Jennifer Doudna |
| author_sort | Martin Jinek |
| collection | DOAJ |
| description | Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells. |
| first_indexed | 2024-04-12T12:15:13Z |
| format | Article |
| id | doaj.art-048712f7a3c54e74a1be3594f16bfea1 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T12:15:13Z |
| publishDate | 2013-01-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-048712f7a3c54e74a1be3594f16bfea12022-12-22T03:33:26ZengeLife Sciences Publications LtdeLife2050-084X2013-01-01210.7554/eLife.00471RNA-programmed genome editing in human cellsMartin Jinek0Alexandra East1Aaron Cheng2Steven Lin3Enbo Ma4Jennifer Doudna5Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesHoward Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesHoward Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; Department of Chemistry, University of California, Berkeley, Berkeley, United States; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, United StatesType II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3′ end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.https://elifesciences.org/articles/00471Cas9endonucleasegenome editing |
| spellingShingle | Martin Jinek Alexandra East Aaron Cheng Steven Lin Enbo Ma Jennifer Doudna RNA-programmed genome editing in human cells eLife Cas9 endonuclease genome editing |
| title | RNA-programmed genome editing in human cells |
| title_full | RNA-programmed genome editing in human cells |
| title_fullStr | RNA-programmed genome editing in human cells |
| title_full_unstemmed | RNA-programmed genome editing in human cells |
| title_short | RNA-programmed genome editing in human cells |
| title_sort | rna programmed genome editing in human cells |
| topic | Cas9 endonuclease genome editing |
| url | https://elifesciences.org/articles/00471 |
| work_keys_str_mv | AT martinjinek rnaprogrammedgenomeeditinginhumancells AT alexandraeast rnaprogrammedgenomeeditinginhumancells AT aaroncheng rnaprogrammedgenomeeditinginhumancells AT stevenlin rnaprogrammedgenomeeditinginhumancells AT enboma rnaprogrammedgenomeeditinginhumancells AT jenniferdoudna rnaprogrammedgenomeeditinginhumancells |