Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening
Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats)...
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| Format: | Article |
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Springer Nature
2017
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| Online Access: | http://hdl.handle.net/1721.1/112748 https://orcid.org/0000-0001-6656-5002 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-7979-3220 https://orcid.org/0000-0003-2782-2509 |
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| author | Joung, Julia Konermann, Silvana M Gootenberg, Jonathan S Abudayyeh, Omar Osama Platt, Randall Jeffrey Brigham, Mark D Sanjana, Neville E Zhang, Feng |
| author2 | Harvard University--MIT Division of Health Sciences and Technology |
| author_facet | Harvard University--MIT Division of Health Sciences and Technology Joung, Julia Konermann, Silvana M Gootenberg, Jonathan S Abudayyeh, Omar Osama Platt, Randall Jeffrey Brigham, Mark D Sanjana, Neville E Zhang, Feng |
| author_sort | Joung, Julia |
| collection | MIT |
| description | Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation. |
| first_indexed | 2024-09-23T14:27:16Z |
| format | Article |
| id | mit-1721.1/112748 |
| institution | Massachusetts Institute of Technology |
| last_indexed | 2024-09-23T14:27:16Z |
| publishDate | 2017 |
| publisher | Springer Nature |
| record_format | dspace |
| spelling | mit-1721.1/1127482022-10-01T21:25:48Z Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening Joung, Julia Konermann, Silvana M Gootenberg, Jonathan S Abudayyeh, Omar Osama Platt, Randall Jeffrey Brigham, Mark D Sanjana, Neville E Zhang, Feng Harvard University--MIT Division of Health Sciences and Technology Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences McGovern Institute for Brain Research at MIT Joung, Julia Konermann, Silvana M Gootenberg, Jonathan S Abudayyeh, Omar Osama Platt, Randall Jeffrey Brigham, Mark D Sanjana, Neville E Zhang, Feng Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation. Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.) McGovern Institute for Brain Research at MIT (Friends of McGovern Institute Fellowship) Massachusetts Institute of Technology. Poitras Center for Affective Disorders Research United States. Department of Energy (Computational Science Graduate Fellowship) National Institute of Mental Health (U.S.) (5DP1-MH100706) National Institute of Mental Health (U.S.) (1R01-MH110049) New York Stem Cell Foundation Poitras Foundation Simons Foundation Paul G. Allen Family Foundation Vallee Foundation Tom Harriman B. Metcalfe 2017-12-14T14:53:35Z 2017-12-14T14:53:35Z 2017-03 2017-12-12T16:16:32Z Article http://purl.org/eprint/type/JournalArticle 1754-2189 1750-2799 http://hdl.handle.net/1721.1/112748 Joung, Julia, et al. “Genome-Scale CRISPR-Cas9 Knockout and Transcriptional Activation Screening.” Nature Protocols, vol. 12, no. 4, Mar. 2017, pp. 828–63. https://orcid.org/0000-0001-6656-5002 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-7979-3220 https://orcid.org/0000-0003-2782-2509 http://dx.doi.org/10.1038/NPROT.2017.016 Nature Protocols Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf Springer Nature PMC |
| spellingShingle | Joung, Julia Konermann, Silvana M Gootenberg, Jonathan S Abudayyeh, Omar Osama Platt, Randall Jeffrey Brigham, Mark D Sanjana, Neville E Zhang, Feng Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening |
| title | Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening |
| title_full | Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening |
| title_fullStr | Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening |
| title_full_unstemmed | Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening |
| title_short | Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening |
| title_sort | genome scale crispr cas9 knockout and transcriptional activation screening |
| url | http://hdl.handle.net/1721.1/112748 https://orcid.org/0000-0001-6656-5002 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0002-7979-3220 https://orcid.org/0000-0003-2782-2509 |
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