Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure

Yan, Qiang; Xu, Kun; Xing, Jiani; Zhang, Tingting; Wang, Xin; Wei, Zehui; Ren, Chonghua; Liu, Zhongtian; Shao, Simin; Zhang, Zhiying

doi:10.1038/srep38970

Cited by 25 publications

(30 citation statements)

References 36 publications

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“…Multiple tRNA-sgRNA units are assembled in a synthetic tRNA-sgRNA cassette, with each sgRNA containing a target-specific spacer, which can be cleaved by endogenous RNase to release mature sgRNAs and tRNA 45 . The sgRNA-shRNA structure hijacks the endogenous shRNA processing system, and multiple sgRNAs and shRNAs are assembled in an sgRNA-shRNA structure format with interval sequences of the Drosha cutting site 46 . Thus, the sgRNA-shRNA transcript is processed into functional sgRNAs and shRNAs by endogenous Drosha.…”

Section: Discussionmentioning

confidence: 99%

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

Zhang

Han

et al. 2017

Molecular Therapy - Nucleic Acids

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Neural cell fate is determined by a tightly controlled transcription regulatory network during development. The ability to manipulate the expression of multiple transcription factors simultaneously is required to delineate the complex picture of neural cell development. Because of the limited carrying capacity of the commonly used viral vectors, such as lentiviral or retroviral vectors, it is often challenging to perform perturbation experiments on multiple transcription factors. Here we have developed a piggyBac (PB) transposon-based CRISPR activation (CRISPRa) all-in-one system, which allows for simultaneous and stable endogenous transactivation of multiple transcription factors and long non-coding RNAs. As a proof of principle, we showed that the PB-CRISPRa system could accelerate the differentiation of human induced pluripotent stem cells into neurons and astrocytes by triggering endogenous expression of different sets of transcription factors. The PB-CRISPRa system has the potential to become a convenient and robust tool in neuroscience, which can meet the needs of a variety of in vitro and in vivo gain-of-function applications.

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Section: Discussionmentioning

confidence: 99%

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

Zhang

Han

et al. 2017

Molecular Therapy - Nucleic Acids

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“…This gRNA processing strategy was later demonstrated in various plant, animal and microbial systems with consistently high efficiencies of multiplex genome editing (Dong et al 2017;Port and Bullock 2016;Shiraki and Kawakami 2018;Tang et al 2019). In addition to the tRNA-gRNA strategy, tandem repeats of gRNA-shRNA (short hairpin RNA) transcripts were also engineered for multiplex genome editing through excision of shRNAs by endogenous ribonuclease DROSHA in mammalian cells (Yan et al 2016).…”

Section: Diverse Strategies For Expressing Multiple Grnasmentioning

confidence: 99%

Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

Hsieh‐Feng

Yang

2020

aBIOTECH

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The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spatiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized transgene-free and multiplex genome editing. In this review, we discuss current strategies and future perspectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.

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“…On the contrary, the use of CRISPR/dCas9 associated with p300 can be used efficiently with a single sgRNA. 20 While CRISPR gene editing was extensively used as a multiplex platform for modifying several genes at the same time, [22][23][24][25][26][27][28][29][30][31][32] only few reports showed the multiplexing capacity of CRISPRa platform, 17,18,33 and none of them in a significant number of genes corresponding to the same cellular lineage.…”

Section: Introductionmentioning

confidence: 99%

Activation of pancreatic β-cell genes by multiplex epigenetic CRISPR-editing

Alejandra

Lucia

et al. 2020

Preprint

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CRISPR-based systems for epigenetic editing are promising molecular tools that could be harnessed for directed differentiation of pluripotent stem cells. We used the CRISPR/dCas9-VP160, CRISPR/dCas9-TET1 and CRISPR/dCas9-P300 systems for multiplex epigenetic editing and activation of human beta pancreatic genes (PDX1, NEUROG3, PAX4 and INS). The CRISPR/dCas9-P300 system was the most effective at activating genes with reduced number of sgRNA. Using small number of sgRNA per gene was important to induce multiplex gene activation. Combined activation of transcription factors (TFs) involved in beta cell development resulted in INS gene expression; in which sequential TFs activation was more effective than simultaneous activation. Full CRISPR RNA-based delivery system was able to activate all targeted genes. Overall, this study shows the utility of CRISPR tools for epigenetic editing and directed cellular differentiation.

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Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure

Cited by 25 publications

References 36 publications

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes

Efficient expression of multiple guide RNAs for CRISPR/Cas genome editing

Activation of pancreatic β-cell genes by multiplex epigenetic CRISPR-editing

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