Single Guide RNA Library Design and Construction
This protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we...
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| Other Authors: | |
| Format: | Article |
| Language: | en_US |
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Cold Spring Harbor Laboratory Press
2016
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| Online Access: | http://hdl.handle.net/1721.1/105776 https://orcid.org/0000-0002-4227-5163 https://orcid.org/0000-0002-1446-7256 |
| _version_ | 1826209246029545472 |
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| author | Wang, Tim Lander, Eric Steven Sabatini, David |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology Wang, Tim Lander, Eric Steven Sabatini, David |
| author_sort | Wang, Tim |
| collection | MIT |
| description | This protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we have developed for both the human and mouse and that are available from the Broad Institute website. Once a set of target genes and corresponding sgRNA sequences has been identified, customized oligonucleotide pools can be rapidly synthesized by a number of commercial vendors. Thereafter, as described here, the oligonucleotides can be efficiently cloned into an appropriate lentiviral expression vector backbone. The resulting plasmid pool can then be packaged into lentiviral particles and used to generate knockouts in any cell line of choice. |
| first_indexed | 2024-09-23T14:19:29Z |
| format | Article |
| id | mit-1721.1/105776 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T14:19:29Z |
| publishDate | 2016 |
| publisher | Cold Spring Harbor Laboratory Press |
| record_format | dspace |
| spelling | mit-1721.1/1057762022-09-29T08:45:05Z Single Guide RNA Library Design and Construction Wang, Tim Lander, Eric Steven Sabatini, David Massachusetts Institute of Technology. Department of Biology Wang, Tim Lander, Eric Steven Sabatini, David This protocol describes how to generate a single guide RNA (sgRNA) library for use in genetic screens. There are many online tools available for predicting sgRNA sequences with high target specificity and/or cleavage activity. Here, we refer the user to genome-wide sgRNA sequence predictions that we have developed for both the human and mouse and that are available from the Broad Institute website. Once a set of target genes and corresponding sgRNA sequences has been identified, customized oligonucleotide pools can be rapidly synthesized by a number of commercial vendors. Thereafter, as described here, the oligonucleotides can be efficiently cloned into an appropriate lentiviral expression vector backbone. The resulting plasmid pool can then be packaged into lentiviral particles and used to generate knockouts in any cell line of choice. 2016-12-09T17:11:51Z 2016-12-09T17:11:51Z 2016-12-09 Article http://purl.org/eprint/type/JournalArticle 1940-3402 1559-6095 http://hdl.handle.net/1721.1/105776 Wang, Tim, Eric S. Lander, and David M. Sabatini. “Single Guide RNA Library Design and Construction.” Cold Spring Harb Protoc 2016, no. 3 (March 2016): pdb.prot090803. https://orcid.org/0000-0002-4227-5163 https://orcid.org/0000-0002-1446-7256 en_US http://dx.doi.org/10.1101/pdb.prot090803 Cold Spring Harbor Protocols Creative Commons Attribution-Noncommercial-Share Alike http://creativecommons.org/licenses/by-nc-sa/4.0/ application/pdf Cold Spring Harbor Laboratory Press PMC |
| spellingShingle | Wang, Tim Lander, Eric Steven Sabatini, David Single Guide RNA Library Design and Construction |
| title | Single Guide RNA Library Design and Construction |
| title_full | Single Guide RNA Library Design and Construction |
| title_fullStr | Single Guide RNA Library Design and Construction |
| title_full_unstemmed | Single Guide RNA Library Design and Construction |
| title_short | Single Guide RNA Library Design and Construction |
| title_sort | single guide rna library design and construction |
| url | http://hdl.handle.net/1721.1/105776 https://orcid.org/0000-0002-4227-5163 https://orcid.org/0000-0002-1446-7256 |
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