CRISPR-mediated transcriptional activation with synthetic guide RNA
| Autor: | Galen W. Miller, Kevin Hemphill, Maren M. Gross, Matthew R. Perkett, Elena Maksimova, Žaklina Strezoska, Eldon T. Chou, Travis Hardcastle, Annaleen Vermeulen, Anja van Brabant Smith, Sarah Michelle Dickerson, Jesse Stombaugh, Emily M. Anderson |
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| Rok vydání: | 2020 |
| Předmět: |
Transcriptional Activation
0106 biological sciences 0301 basic medicine Bioengineering Computational biology Biology 01 natural sciences Applied Microbiology and Biotechnology Mice 03 medical and health sciences 010608 biotechnology Animals Humans CRISPR Guide RNA Gene Gene Editing Regulation of gene expression Trans-activating crRNA Messenger RNA Cas9 General Medicine Aptamers Nucleotide HEK293 Cells 030104 developmental biology NIH 3T3 Cells Cytokine secretion CRISPR-Cas Systems RNA Guide Kinetoplastida Biotechnology |
| Zdroj: | Journal of Biotechnology. 319:25-35 |
| ISSN: | 0168-1656 |
| DOI: | 10.1016/j.jbiotec.2020.05.005 |
| Popis: | The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format. |
| Databáze: | OpenAIRE |
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