Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells
The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockou...
| Main Authors: | , , , , , , , , , , |
|---|---|
| Other Authors: | |
| Format: | Article |
| Language: | en_US |
| Published: |
American Association for the Advancement of Science (AAAS)
2017
|
| Online Access: | http://hdl.handle.net/1721.1/111576 https://orcid.org/0000-0002-2639-9879 https://orcid.org/0000-0003-2782-2509 |
| _version_ | 1811094808885723136 |
|---|---|
| author | Hartenian, E. Shi, X. Mikkelsen, T. S. Heckl, D. Ebert, B. L. Root, D. E. Doench, J. G. Shalem, Ophir Sanjana, Neville E Scott, David Arthur Zhang, Feng |
| author2 | McGovern Institute for Brain Research at MIT |
| author_facet | McGovern Institute for Brain Research at MIT Hartenian, E. Shi, X. Mikkelsen, T. S. Heckl, D. Ebert, B. L. Root, D. E. Doench, J. G. Shalem, Ophir Sanjana, Neville E Scott, David Arthur Zhang, Feng |
| author_sort | Hartenian, E. |
| collection | MIT |
| description | The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9. |
| first_indexed | 2024-09-23T16:05:28Z |
| format | Article |
| id | mit-1721.1/111576 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T16:05:28Z |
| publishDate | 2017 |
| publisher | American Association for the Advancement of Science (AAAS) |
| record_format | dspace |
| spelling | mit-1721.1/1115762022-10-02T06:17:41Z Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells Hartenian, E. Shi, X. Mikkelsen, T. S. Heckl, D. Ebert, B. L. Root, D. E. Doench, J. G. Shalem, Ophir Sanjana, Neville E Scott, David Arthur Zhang, Feng McGovern Institute for Brain Research at MIT Shalem, Ophir Sanjana, Neville E Scott, David Arthur Zhang, Feng The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats)–associated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9. National Institutes of Health (U.S.) (Award 1DP1-MH100706) National Institutes of Health (U.S.) (1R01-DK097768) 2017-09-15T20:18:35Z 2017-09-15T20:18:35Z 2014-01 2013-10 Article http://purl.org/eprint/type/JournalArticle 0036-8075 1095-9203 http://hdl.handle.net/1721.1/111576 Shalem, O. "Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells." Science 343, 6166 (January 2014): 84-87 © 2014 American Association for the Advancement of Science (AAAS) https://orcid.org/0000-0002-2639-9879 https://orcid.org/0000-0003-2782-2509 en_US http://dx.doi.org/10.1126/science.1247005 Science Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf American Association for the Advancement of Science (AAAS) PMC |
| spellingShingle | Hartenian, E. Shi, X. Mikkelsen, T. S. Heckl, D. Ebert, B. L. Root, D. E. Doench, J. G. Shalem, Ophir Sanjana, Neville E Scott, David Arthur Zhang, Feng Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells |
| title | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells |
| title_full | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells |
| title_fullStr | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells |
| title_full_unstemmed | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells |
| title_short | Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells |
| title_sort | genome scale crispr cas9 knockout screening in human cells |
| url | http://hdl.handle.net/1721.1/111576 https://orcid.org/0000-0002-2639-9879 https://orcid.org/0000-0003-2782-2509 |
| work_keys_str_mv | AT harteniane genomescalecrisprcas9knockoutscreeninginhumancells AT shix genomescalecrisprcas9knockoutscreeninginhumancells AT mikkelsents genomescalecrisprcas9knockoutscreeninginhumancells AT heckld genomescalecrisprcas9knockoutscreeninginhumancells AT ebertbl genomescalecrisprcas9knockoutscreeninginhumancells AT rootde genomescalecrisprcas9knockoutscreeninginhumancells AT doenchjg genomescalecrisprcas9knockoutscreeninginhumancells AT shalemophir genomescalecrisprcas9knockoutscreeninginhumancells AT sanjananevillee genomescalecrisprcas9knockoutscreeninginhumancells AT scottdavidarthur genomescalecrisprcas9knockoutscreeninginhumancells AT zhangfeng genomescalecrisprcas9knockoutscreeninginhumancells |