Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery
The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifical...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2014-12-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/04766 |
| _version_ | 1828375131061223424 |
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| author | Steven Lin Brett T Staahl Ravi K Alla Jennifer A Doudna |
| author_facet | Steven Lin Brett T Staahl Ravi K Alla Jennifer A Doudna |
| author_sort | Steven Lin |
| collection | DOAJ |
| description | The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells. |
| first_indexed | 2024-04-14T07:42:15Z |
| format | Article |
| id | doaj.art-827b3586ab1e43eba0b9099d5b77271b |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-14T07:42:15Z |
| publishDate | 2014-12-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-827b3586ab1e43eba0b9099d5b77271b2022-12-22T02:05:26ZengeLife Sciences Publications LtdeLife2050-084X2014-12-01310.7554/eLife.04766Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 deliverySteven Lin0Brett T Staahl1Ravi K Alla2Jennifer A Doudna3Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United StatesComputational Genomics Resource Laboratory, QB3, University of California, Berkeley, Berkeley, United StatesDepartment of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States; Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States; Department of Chemistry, University of California, Berkeley, Berkeley, United States; Department of Chemistry, Lawrence Berkeley National Laboratory, Berkeley, United StatesThe CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.https://elifesciences.org/articles/04766CRISPR/Cas9genome engineeringhomologous recombinationcell cycle synchronizationnocodazolenon-homologous end joining |
| spellingShingle | Steven Lin Brett T Staahl Ravi K Alla Jennifer A Doudna Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery eLife CRISPR/Cas9 genome engineering homologous recombination cell cycle synchronization nocodazole non-homologous end joining |
| title | Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery |
| title_full | Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery |
| title_fullStr | Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery |
| title_full_unstemmed | Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery |
| title_short | Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery |
| title_sort | enhanced homology directed human genome engineering by controlled timing of crispr cas9 delivery |
| topic | CRISPR/Cas9 genome engineering homologous recombination cell cycle synchronization nocodazole non-homologous end joining |
| url | https://elifesciences.org/articles/04766 |
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