Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging
Abstract CRISPR/dCas9 binds precisely to defined genomic sequences through targeting of guide RNA (gRNA) sequences. In vivo imaging of genomic loci can be achieved by recruiting fluorescent proteins using either dCas9 or gRNA. We thoroughly validate and compare the effectiveness and specificity of s...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2018-03-01
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| Series: | Genome Biology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13059-018-1413-5 |
| _version_ | 1818480385086980096 |
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| author | Yu Hong Guangqing Lu Jinzhi Duan Wenjing Liu Yu Zhang |
| author_facet | Yu Hong Guangqing Lu Jinzhi Duan Wenjing Liu Yu Zhang |
| author_sort | Yu Hong |
| collection | DOAJ |
| description | Abstract CRISPR/dCas9 binds precisely to defined genomic sequences through targeting of guide RNA (gRNA) sequences. In vivo imaging of genomic loci can be achieved by recruiting fluorescent proteins using either dCas9 or gRNA. We thoroughly validate and compare the effectiveness and specificity of several dCas9/gRNA genome labeling systems. Surprisingly, we discover that in the gRNA-labeling strategies, accumulation of tagged gRNA transcripts leads to non-specific labeling foci. Furthermore, we develop novel bimolecular fluorescence complementation (BIFC) methods that combine the advantages of both dCas9-labeling and gRNA-labeling strategies. The BIFC-dCas9/gRNA methods demonstrate high signal-to-noise ratios and have no non-specific foci. |
| first_indexed | 2024-12-10T11:22:48Z |
| format | Article |
| id | doaj.art-06562463dc93442b961ea9d99b7dd3e6 |
| institution | Directory Open Access Journal |
| issn | 1474-760X |
| language | English |
| last_indexed | 2024-12-10T11:22:48Z |
| publishDate | 2018-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Genome Biology |
| spelling | doaj.art-06562463dc93442b961ea9d99b7dd3e62022-12-22T01:50:51ZengBMCGenome Biology1474-760X2018-03-0119111010.1186/s13059-018-1413-5Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imagingYu Hong0Guangqing Lu1Jinzhi Duan2Wenjing Liu3Yu Zhang4Peking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Peking UniversityGraduate School of Peking Union Medical CollegeGraduate School of Peking Union Medical CollegeGraduate School of Peking Union Medical CollegePeking University-Tsinghua University-National Institute of Biological Sciences Joint Graduate Program, School of Life Sciences, Peking UniversityAbstract CRISPR/dCas9 binds precisely to defined genomic sequences through targeting of guide RNA (gRNA) sequences. In vivo imaging of genomic loci can be achieved by recruiting fluorescent proteins using either dCas9 or gRNA. We thoroughly validate and compare the effectiveness and specificity of several dCas9/gRNA genome labeling systems. Surprisingly, we discover that in the gRNA-labeling strategies, accumulation of tagged gRNA transcripts leads to non-specific labeling foci. Furthermore, we develop novel bimolecular fluorescence complementation (BIFC) methods that combine the advantages of both dCas9-labeling and gRNA-labeling strategies. The BIFC-dCas9/gRNA methods demonstrate high signal-to-noise ratios and have no non-specific foci.http://link.springer.com/article/10.1186/s13059-018-1413-5Genome labelingCRISPR/dCas9Bimolecular fluorescence complementation (BIFC) |
| spellingShingle | Yu Hong Guangqing Lu Jinzhi Duan Wenjing Liu Yu Zhang Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging Genome Biology Genome labeling CRISPR/dCas9 Bimolecular fluorescence complementation (BIFC) |
| title | Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging |
| title_full | Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging |
| title_fullStr | Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging |
| title_full_unstemmed | Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging |
| title_short | Comparison and optimization of CRISPR/dCas9/gRNA genome-labeling systems for live cell imaging |
| title_sort | comparison and optimization of crispr dcas9 grna genome labeling systems for live cell imaging |
| topic | Genome labeling CRISPR/dCas9 Bimolecular fluorescence complementation (BIFC) |
| url | http://link.springer.com/article/10.1186/s13059-018-1413-5 |
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