Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems

Lowder, Levi G.; Paul, Joel R.; Qi, Yiping

doi:10.1007/978-1-4939-7125-1_12

Cited by 59 publications

(46 citation statements)

References 26 publications

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“…The transcriptional effectors enable RNA polymerase and cofactor recruitment for regulation of gene expression (36). Lowder et al (37) developed a protocol for application of VP64, as described above, for activation of plant gene expression and SRDX for repression.…”

Section: Expanding the Crispr/cas Toolboxmentioning

confidence: 99%

Applying gene editing to tailor precise genetic modifications in plants

Eck

2020

Journal of Biological Chemistry

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The ability to tailor alterations in genomes, including plant genomes, in a site-specific manner has been greatly advanced through approaches that reduced the complexity and time of genome sequencing along with development of gene editing technologies. These technologies provide a valuable foundation for studies of gene function, metabolic engineering and trait modification for crop improvement. Development of genome editing methodologies began approximately 20 years ago first with meganucleases followed by Zinc Finger Nucleases, Transcriptional Activator-Like Effector Nucleases and, most recently, Clustered Regulatory Interspaced Short Palindromic Repeat (CRISPR)-Associated Protein (Cas) (CRISPR/Cas), which is by far the most utilized method. The premise of CRISPR/Cas centers on the cleaving of one or both DNA strands by a Cas protein, an endonuclease, followed by mending of the DNA by repair mechanisms inherent in cells. Its user-friendly construct design, greater flexibility in targeting genomic regions, and the cost-effective attributes have resulted in it being widely adopted and revolutionizing precise modification of the genomes of many organisms. Indeed, the CRISPR/Cas system has been utilized for gene editing in many plant species including important food crops such as maize, wheat, rice, and potatoes. This review summarizes the various approaches including the most recent designs being used to make modifications from as small as a single base pair change to insertion of DNA fragments. On the gene expression level, strategies are presented that make it possible to knock-out or modulate through activation and repression. Also discussed are pre-requisites necessary for CRISPR/Cas-mediated editing as well as the current challenges.

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Section: Expanding the Crispr/cas Toolboxmentioning

confidence: 99%

Applying gene editing to tailor precise genetic modifications in plants

Eck

2020

Journal of Biological Chemistry

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“…The dCas9 is incapable of cleaving any DNA but is successfully guided to the promoter sequence of desirable MIR genes. In contrast, it can be fused to other functional domains, for example dCas9:VP64 (quadruple tandem repeat of the Herpes simplex virus VP16‐activation domain), dCas9:SRDX (synthetic transcriptional repressor pco‐dCas9‐3X) or dCas9:SET (methyltransferase domain of the H3K9me3 writer) and dCas9:AT (acetyltransferase domain), which act as transcriptional activators (Chavez et al ., ), repressors (Lowder et al ., ) or epigenetic modifiers (O'Geen et al ., ), respectively. The transcriptional modulation starts by dCas9 (or dCfp1) guided by gRNA to sequences immediately upstream of the transcriptional start site (TSS) of an MIR gene.…”

Section: Biotechnological Approaches To Fine‐tuning Of Mirna Activitymentioning

confidence: 99%

MicroRNAs and new biotechnological tools for its modulation and improving stress tolerance in plants

Basso

Ferreira

Kobayashi

et al. 2019

Plant Biotechnology Journal

118

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Summary Micro RNA s (mi RNA s) modulate the abundance and spatial–temporal accumulation of target mRNA s and indirectly regulate several plant processes. Transcriptional regulation of the genes encoding mi RNA s ( MIR genes) can be activated by numerous transcription factors, which themselves are regulated by other mi RNA s. Fine‐tuning of MIR genes or mi RNA s is a powerful biotechnological strategy to improve tolerance to abiotic or biotic stresses in crops of economic importance. Current approaches for mi RNA fine‐tuning are based on the down‐ or up‐regulation of MIR gene transcription and the use of genetic engineering tools to manipulate the final concentration of these mi RNA s in the cytoplasm. Transgenesis, cisgenesis, intragenesis, artificial MIR genes, endogenous and artificial target mimicry, MIR genes editing using Meganucleases, ZNF proteins, TALEN s and CRISPR /Cas9 or CRISPR /Cpf1, CRISPR / dC as9 or dC pf1, CRISPR 13a, topical delivery of mi RNA s and epigenetic memory have been successfully explored to MIR gene or mi RNA modulation and improve agronomic traits in several model or crop plants. However, advantages and drawbacks of each of these new biotechnological tools ( NBT s) are still not well understood. In this review, we provide a brief overview of the biogenesis and role of mi RNA s in response to abiotic or biotic stresses, we present critically the main NBT s used for the manipulation of MIR genes and mi RNA s, we show current efforts and findings with the MIR genes and mi RNA s modulation in plants, and we summarize the advantages and drawbacks of these NBT s and provide some alternatives to overcome. Finally, challenges and future perspectives to mi RNA modulating in important crops are also discussed.

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“…The CRISPR/dCas9 can be used as a platform to regulate gene expression in plants, as well. Transcription effectors such as VP64 and SRDX can be fused to dCas9 and lead to activation or deactivation of the targeted gene, respectively [101]. Piatek et al (2015) examined the ability of the dCas9 fused to EDLL (transcription activator) and SRDX (transcription repressor) to regulate transcription of the target gene.…”

Section: Strategies For Reducing Off-target Mutationsmentioning

confidence: 99%

Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

Hajiahmadi

Movahedi

Wei

et al. 2019

IJMS

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The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

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Multiplexed Transcriptional Activation or Repression in Plants Using CRISPR-dCas9-Based Systems

Cited by 59 publications

References 26 publications

Applying gene editing to tailor precise genetic modifications in plants

Applying gene editing to tailor precise genetic modifications in plants

MicroRNAs and new biotechnological tools for its modulation and improving stress tolerance in plants

Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

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