Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila
The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster. This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance th...
| Main Authors: | , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Oxford University Press
2020-12-01
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| Series: | G3: Genes, Genomes, Genetics |
| Subjects: | |
| Online Access: | http://g3journal.org/lookup/doi/10.1534/g3.120.401614 |
| _version_ | 1818367756543721472 |
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| author | Decai Mao Yu Jia Ping Peng Da Shen Xingjie Ren Ruibao Zhu Yuhao Qiu Yuting Han Jinchao Yu Qinyun Che Yutong Li Xinyi Lu Lu-Ping Liu Zhao Wang Qingfei Liu Jin Sun Jian-Quan Ni |
| author_facet | Decai Mao Yu Jia Ping Peng Da Shen Xingjie Ren Ruibao Zhu Yuhao Qiu Yuting Han Jinchao Yu Qinyun Che Yutong Li Xinyi Lu Lu-Ping Liu Zhao Wang Qingfei Liu Jin Sun Jian-Quan Ni |
| author_sort | Decai Mao |
| collection | DOAJ |
| description | The flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster. This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo. In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies. |
| first_indexed | 2024-12-13T22:57:07Z |
| format | Article |
| id | doaj.art-a3a0739dd0ef4c81a5e64dae6bf322e2 |
| institution | Directory Open Access Journal |
| issn | 2160-1836 |
| language | English |
| last_indexed | 2024-12-13T22:57:07Z |
| publishDate | 2020-12-01 |
| publisher | Oxford University Press |
| record_format | Article |
| series | G3: Genes, Genomes, Genetics |
| spelling | doaj.art-a3a0739dd0ef4c81a5e64dae6bf322e22022-12-21T23:28:29ZengOxford University PressG3: Genes, Genomes, Genetics2160-18362020-12-0110124483448810.1534/g3.120.40161418Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in DrosophilaDecai MaoYu JiaPing PengDa ShenXingjie RenRuibao ZhuYuhao QiuYuting HanJinchao YuQinyun CheYutong LiXinyi LuLu-Ping LiuZhao WangQingfei LiuJin SunJian-Quan NiThe flySAM/CRISPRa system has recently emerged as a powerful tool for gain-of-function studies in Drosophila melanogaster. This system includes Gal4/UAS-driven dCas9 activators and U6 promoter-controlled sgRNA. Having established dCas9 activators superior to other combinations, to further enhance the efficiency of the targeting activators we systematically optimized the parameters of the sgRNA. Interestingly, the most efficient sgRNAs were found to accumulate in the region from -150bp to -450bp upstream of the transcription start site (TSS), and the activation efficiency showed a strong positive correlation with the GC content of the sgRNA targeting sequence. In addition, the target region is dominant to the GC content, as sgRNAs targeting areas beyond -600bp from the TSS lose efficiency even when containing 75% GC. Surprisingly, when comparing the activities of sgRNAs targeting to either DNA strand, sgRNAs targeting to the non-template strand outperform those complementary to the template strand, both in cells and in vivo. In summary, we define criteria for sgRNA design which will greatly facilitate the application of CRISPRa in gain-of-function studies.http://g3journal.org/lookup/doi/10.1534/g3.120.401614flysamsgrnaposition effectgc contentdna strand |
| spellingShingle | Decai Mao Yu Jia Ping Peng Da Shen Xingjie Ren Ruibao Zhu Yuhao Qiu Yuting Han Jinchao Yu Qinyun Che Yutong Li Xinyi Lu Lu-Ping Liu Zhao Wang Qingfei Liu Jin Sun Jian-Quan Ni Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila G3: Genes, Genomes, Genetics flysam sgrna position effect gc content dna strand |
| title | Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila |
| title_full | Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila |
| title_fullStr | Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila |
| title_full_unstemmed | Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila |
| title_short | Enhanced Efficiency of flySAM by Optimization of sgRNA Parameters in Drosophila |
| title_sort | enhanced efficiency of flysam by optimization of sgrna parameters in drosophila |
| topic | flysam sgrna position effect gc content dna strand |
| url | http://g3journal.org/lookup/doi/10.1534/g3.120.401614 |
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