Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening
Abstract Background Large-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biologic...
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| Format: | Article |
| Language: | English |
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BMC
2019-03-01
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| Series: | BMC Genomics |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12864-019-5601-9 |
| _version_ | 1818775111246807040 |
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| author | Ning Sun Sakina Petiwala Rui Wang Charles Lu Mufeng Hu Sujana Ghosh Yan Hao Christopher P. Miller Namjin Chung |
| author_facet | Ning Sun Sakina Petiwala Rui Wang Charles Lu Mufeng Hu Sujana Ghosh Yan Hao Christopher P. Miller Namjin Chung |
| author_sort | Ning Sun |
| collection | DOAJ |
| description | Abstract Background Large-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. Here, we report the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control. Results We designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for a tight and inducible control of gene knockout in vitro, and in vivo during a seven-week-long experiment following hematopoietic reconstitution in mice. We next performed parallel genome-wide loss-of-function screens using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, we observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, we demonstrate minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings. Conclusions In this study, we have developed a drug-inducible CRISPR-Cas9 system that shows high cleavage efficiency upon induction but low background activity. Using this system, we have achieved inducible gene disruption in a wide range of cell types both in vitro and in vivo. For the first time, we present a systematic side-by-side comparison of constitutive and drug-inducible CRISPR-Cas9 platforms in large-scale functional screens. We demonstrate the tightness and efficiency of our drug-inducible CRISPR-Cas9 system in genome-wide pooled screening. Our design increases the versatility of CRISPR-based genetic screening and represents a significant upgrade on existing functional genomics toolbox. |
| first_indexed | 2024-12-18T10:51:50Z |
| format | Article |
| id | doaj.art-4b39644012e04178a8ae8b80c1b57d95 |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-12-18T10:51:50Z |
| publishDate | 2019-03-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-4b39644012e04178a8ae8b80c1b57d952022-12-21T21:10:26ZengBMCBMC Genomics1471-21642019-03-0120111510.1186/s12864-019-5601-9Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screeningNing Sun0Sakina Petiwala1Rui Wang2Charles Lu3Mufeng Hu4Sujana Ghosh5Yan Hao6Christopher P. Miller7Namjin Chung8AbbVie Inc.AbbVie Inc.AbbVie Cambridge Research CenterAbbVie Inc.AbbVie Inc.AbbVie Inc.AbbVie Inc.AbbVie Inc.AbbVie Inc.Abstract Background Large-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. Here, we report the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control. Results We designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for a tight and inducible control of gene knockout in vitro, and in vivo during a seven-week-long experiment following hematopoietic reconstitution in mice. We next performed parallel genome-wide loss-of-function screens using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, we observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, we demonstrate minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings. Conclusions In this study, we have developed a drug-inducible CRISPR-Cas9 system that shows high cleavage efficiency upon induction but low background activity. Using this system, we have achieved inducible gene disruption in a wide range of cell types both in vitro and in vivo. For the first time, we present a systematic side-by-side comparison of constitutive and drug-inducible CRISPR-Cas9 platforms in large-scale functional screens. We demonstrate the tightness and efficiency of our drug-inducible CRISPR-Cas9 system in genome-wide pooled screening. Our design increases the versatility of CRISPR-based genetic screening and represents a significant upgrade on existing functional genomics toolbox.http://link.springer.com/article/10.1186/s12864-019-5601-9CRISPRGene editingFunctional genomics |
| spellingShingle | Ning Sun Sakina Petiwala Rui Wang Charles Lu Mufeng Hu Sujana Ghosh Yan Hao Christopher P. Miller Namjin Chung Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening BMC Genomics CRISPR Gene editing Functional genomics |
| title | Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening |
| title_full | Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening |
| title_fullStr | Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening |
| title_full_unstemmed | Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening |
| title_short | Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening |
| title_sort | development of drug inducible crispr cas9 systems for large scale functional screening |
| topic | CRISPR Gene editing Functional genomics |
| url | http://link.springer.com/article/10.1186/s12864-019-5601-9 |
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