Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways

Dai, Zhen; Wu, Rong; Zhao, Yicheng; Wang, Kankan; Huang, Yongye; Xie, Zhixun; Tu, Changchun; Ouyang, Hongsheng; Wang, Tiedong; Pang, Daxin

doi:10.1631/jzus.b1400001

Cited by 11 publications

(9 citation statements)

References 18 publications

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“…However, small interfering RNAs (siRNAs) target mRNA transcripts and do not target viral DNA in the host cells. Moreover, stable expression of short hairpin RNA (shRNA) is prone to cellular toxicity by overloading the native miRNA pathway with shRNAs [ 15 , 16 ]. The CRISPR/Cas9 system is an adaptive innate immune system that provides resistance to microbes by targeting the nucleic acid of invading bacteriophages in a sequence-specific manner [ 17 ].…”

Section: Introductionmentioning

confidence: 99%

In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

et al. 2021

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Marek’s disease (MD), caused by MD herpesvirus (MDV), is an economically important disease in chickens. The efficacy of the existing vaccines against evolving virulent stains may become limited and necessitates the development of novel antiviral strategies to protect poultry from MDV strains with increased virulence. The CRISPR/Cas9 system has emerged as a powerful genome editing tool providing an opportunity to develop antiviral strategies for the control of MDV infection. Here, we characterized Tol2 transposon constructs encoding Cas9 and guide RNAs (gRNAs) specific to the immediate early infected-cell polypeptide-4 (ICP4) of MDV. We generated transgenic chickens that constitutively express Cas9 and ICP4-gRNAs (gICP4) and challenged them via intraabdominal injection of MDV-1 Woodlands strain passage-19 (p19). Transgenic chickens expressing both gRNA/Cas9 had a significantly reduced replication of MDV in comparison to either transgenic Cas9-only or the wild-type (WT) chickens. We further confirmed that the designed gRNAs exhibited sequence-specific virus interference in transgenic chicken embryo fibroblast (CEF) expressing Cas9/gICP4 when infected with MDV but not with herpesvirus of turkeys (HVT). These results suggest that CRISPR/Cas9 can be used as an antiviral approach to control MDV infection in chickens, allowing HVT to be used as a vector for recombinant vaccines.

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Section: Introductionmentioning

confidence: 99%

In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

et al. 2021

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“…For example, one of the greatest limitations of using miRNAs as therapeutic targets is the high number of off-targets [47] and undesirable side effects [48] of their introduction, even leading to cell death in drastic cases [49]. It is likely that non-canonical interactions are partially responsible for those effects.…”

Section: Discussionmentioning

confidence: 99%

Understanding the Modus Operandi of MicroRNA Regulatory Clusters

Oliveira

Bovolenta

Alves

et al. 2019

Cells

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MicroRNAs (miRNAs) are non-coding RNAs that regulate a wide range of biological pathways by post-transcriptionally modulating gene expression levels. Given that even a single miRNA may simultaneously control several genes enrolled in multiple biological functions, one would expect that these tiny RNAs have the ability to properly sort among distinctive cellular processes to drive protein production. To test this hypothesis, we scrutinized previously published microarray datasets and clustered protein-coding gene expression profiles according to the intensity of fold-change levels caused by the exogenous transfection of 10 miRNAs (miR-1, miR-7, miR-9, miR-124, miR-128a, miR-132, miR-133a, miR-142, miR-148b, miR-181a) in a human cell line. Through an in silico functional enrichment analysis, we discovered non-randomic regulatory patterns, proper of each cluster identified. We demonstrated that miRNAs are capable of equivalently modulate the expression signatures of target genes in regulatory clusters according to the biological function they are assigned to. Moreover, target prediction analysis applied to ten vertebrate species, suggest that such miRNA regulatory modus operandi is evolutionarily conserved within vertebrates. Overall, we discovered a complex regulatory cluster-module strategy driven by miRNAs, which relies on the controlled intensity of the repression over distinct targets under specific biological contexts. Our discovery helps to clarify the mechanisms underlying the functional activity of miRNAs and makes it easier to take the fastest and most accurate path in the search for the functions of miRNAs in any distinct biological process of interest.

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“…This has also been observed in transgenic pigs expressing anti-classical swine fever virus shRNA (Dai et al 2014). This can be overcome by decreasing the strength of the promoter, incorporating the shRNA into an endogenous miRNA intron or using tissue-specific or transducible promoters (Furth et al 1994;Dickins et al 2007).…”

Section: Technologymentioning

confidence: 99%

RNA interference-based technology: what role in animal agriculture?

et al. 2017

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Abstract. Animal agriculture faces a broad array of challenges, ranging from disease threats to adverse environmental conditions, while attempting to increase productivity using fewer resources. RNA interference (RNAi) is a biological phenomenon with the potential to provide novel solutions to some of these challenges. Discovered just 20 years ago, the mechanisms underlying RNAi are now well described in plants and animals. Intracellular double-stranded RNA triggers a conserved response that leads to cleavage and degradation of complementary mRNA strands, thereby preventing production of the corresponding protein product. RNAi can be naturally induced by expression of endogenous microRNA, which are critical in the regulation of protein synthesis, providing a mechanism for rapid adaptation of physiological function. This endogenous pathway can be co-opted for targeted RNAi either through delivery of exogenous small interfering RNA (siRNA) into target cells or by transgenic expression of short hairpin RNA (shRNA). Potentially valuable RNAi targets for livestock include endogenous genes such as developmental regulators, transcripts involved in adaptations to new physiological states, immune response mediators, and also exogenous genes such as those encoded by viruses. RNAi approaches have shown promise in cell culture and rodent models as well as some livestock studies, but technical and market barriers still need to be addressed before commercial applications of RNAi in animal agriculture can be realised. Key challenges for exogenous delivery of siRNA include appropriate formulation for physical delivery, internal transport and eventual cellular uptake of the siRNA; additionally, rigorous safety and residue studies in target species will be necessary for siRNA delivery nanoparticles currently under evaluation. However, genomic incorporation of shRNA can overcome these issues, but optimal promoters to drive shRNA expression are needed, and genetic engineering may attract more resistance from consumers than the use of exogenous siRNA. Despite these hurdles, the convergence of greater understanding of RNAi mechanisms, detailed descriptions of regulatory processes in animal development and disease, and breakthroughs in synthetic chemistry and genome engineering has created exciting possibilities for using RNAi to enhance the sustainability of animal agriculture.

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Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways

Cited by 11 publications

References 18 publications

In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

In Vivo Inhibition of Marek’s Disease Virus in Transgenic Chickens Expressing Cas9 and gRNA against ICP4

Understanding the Modus Operandi of MicroRNA Regulatory Clusters

RNA interference-based technology: what role in animal agriculture?

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