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Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript
Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci en...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10218229/ https://www.ncbi.nlm.nih.gov/pubmed/37239880 http://dx.doi.org/10.3390/ijms24108535 |
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author | Jiang, Chaoqian Geng, Lishuang Wang, Jinpeng Liang, Yingjuan Guo, Xiaochen Liu, Chang Zhao, Yunjing Jin, Junxue Liu, Zhonghua Mu, Yanshuang |
author_facet | Jiang, Chaoqian Geng, Lishuang Wang, Jinpeng Liang, Yingjuan Guo, Xiaochen Liu, Chang Zhao, Yunjing Jin, Junxue Liu, Zhonghua Mu, Yanshuang |
author_sort | Jiang, Chaoqian |
collection | PubMed |
description | Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem–loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications. |
format | Online Article Text |
id | pubmed-10218229 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-102182292023-05-27 Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript Jiang, Chaoqian Geng, Lishuang Wang, Jinpeng Liang, Yingjuan Guo, Xiaochen Liu, Chang Zhao, Yunjing Jin, Junxue Liu, Zhonghua Mu, Yanshuang Int J Mol Sci Article Simultaneously, multiplexed genome engineering and targeting multiple genomic loci are valuable to elucidating gene interactions and characterizing genetic networks that affect phenotypes. Here, we developed a general CRISPR-based platform to perform four functions and target multiple genome loci encoded in a single transcript. To establish multiple functions for multiple loci targets, we fused four RNA hairpins, MS2, PP7, com and boxB, to stem–loops of gRNA (guide RNA) scaffolds, separately. The RNA-hairpin-binding domains MCP, PCP, Com and λN22 were fused with different functional effectors. These paired combinations of cognate-RNA hairpins and RNA-binding proteins generated the simultaneous, independent regulation of multiple target genes. To ensure that all proteins and RNAs are expressed in one transcript, multiple gRNAs were constructed in a tandemly arrayed tRNA (transfer RNA)-gRNA architecture, and the triplex sequence was cloned between the protein-coding sequences and the tRNA-gRNA array. By leveraging this system, we illustrate the transcriptional activation, transcriptional repression, DNA methylation and DNA demethylation of endogenous targets using up to 16 individual CRISPR gRNAs delivered on a single transcript. This system provides a powerful platform to investigate synthetic biology questions and engineer complex-phenotype medical applications. MDPI 2023-05-10 /pmc/articles/PMC10218229/ /pubmed/37239880 http://dx.doi.org/10.3390/ijms24108535 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Jiang, Chaoqian Geng, Lishuang Wang, Jinpeng Liang, Yingjuan Guo, Xiaochen Liu, Chang Zhao, Yunjing Jin, Junxue Liu, Zhonghua Mu, Yanshuang Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript |
title | Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript |
title_full | Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript |
title_fullStr | Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript |
title_full_unstemmed | Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript |
title_short | Multiplexed Gene Engineering Based on dCas9 and gRNA-tRNA Array Encoded on Single Transcript |
title_sort | multiplexed gene engineering based on dcas9 and grna-trna array encoded on single transcript |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10218229/ https://www.ncbi.nlm.nih.gov/pubmed/37239880 http://dx.doi.org/10.3390/ijms24108535 |
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