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Method Article
Yangming Wang
Yangming's Lab Peking University
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
microRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in plants, animals and viruses. However, measuring miRNA activity in vivo remains a big challenge. In this protocol, using a miRNA-mediated sgRNA releasing strategy and dCas9-VPR to drive a transgene RFP expression, we create a miRNA sensor that can faithfully measure miRNA activity at cellular levels. When sgRNAs are designed to target endogenous locus, we show this system can be adapted to achieve cell type specific activation of endogenous genes. Furthermore, when dCas9 is fused with a transcriptional repressor or a base editor, we show this system can be used to repress the expression of endogenous genes or mutate specific DNA bases of chromosome upon induction by cell type-specific miRNAs. This step-by-step protocol is related to the publication “A microRNA-inducible CRISPR-Cas9 platform serves as microRNA sensors and cell type specific genome regulation tools” in Nature Cell Biology.
Keywords
microRNA, CRISPR-Cas9, CRISPR-on, CRISPRi, Base editing, Genome editing
Figure 1
Two patent applications have been filed relating to this protocol.
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Method Article
Yangming Wang
Yangming's Lab Peking University
Corresponding Author
Version History
CURRENT STATUS: Posted
Version 1
Posted 25 Feb, 2019
No community comments so far
Metrics
Comments: 0
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
microRNAs (miRNAs) are small noncoding RNAs that play important regulatory roles in plants, animals and viruses. However, measuring miRNA activity in vivo remains a big challenge. In this protocol, using a miRNA-mediated sgRNA releasing strategy and dCas9-VPR to drive a transgene RFP expression, we create a miRNA sensor that can faithfully measure miRNA activity at cellular levels. When sgRNAs are designed to target endogenous locus, we show this system can be adapted to achieve cell type specific activation of endogenous genes. Furthermore, when dCas9 is fused with a transcriptional repressor or a base editor, we show this system can be used to repress the expression of endogenous genes or mutate specific DNA bases of chromosome upon induction by cell type-specific miRNAs. This step-by-step protocol is related to the publication “A microRNA-inducible CRISPR-Cas9 platform serves as microRNA sensors and cell type specific genome regulation tools” in Nature Cell Biology.
Figure 1
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