Efficient Gene Knockout in Goats Using CRISPR/Cas9 System

Ni, Wei; Qiao, Jun; Hu, Shengwei; Zhao, Xinxia; Regouski, Misha; Yang, Min; Polejaeva, Irina A.; Chen, Chuangfu

doi:10.1371/journal.pone.0106718

Cited by 210 publications

(142 citation statements)

References 35 publications

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“…Recently, gene editing has been applied to many species, some of which are used to produce agricultural products. [1][2][3][4][5][6][7][8][9][10] Regarding techniques for gene editing, the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system is a much easier approach than the use of transcription activator-like effector nuclease (TALEN) and zinc finger nuclease (ZFN). This has led to the CRISPR/Cas9 system attracting substantial attention as a new tool for gene editing.…”

mentioning

confidence: 99%

“…This has led to the CRISPR/Cas9 system attracting substantial attention as a new tool for gene editing. [3][4][5][6][7][8][9][10] Unfortunately, it is associated with the problem of inducing off-target mutations; however, to the best of our knowledge, no studies have comprehensively investigated GMOs produced by this system for off-target mutations. It is anticipated that this problem will soon be reduced or resolved because the CRISPR/Cas9 system is continually being improved.…”

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confidence: 99%

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Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy

Nakajima

Nishimaki-Mogami

Kondo

2016

Biological & Pharmaceutical Bulletin

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Genome editing has undergone rapid development during the last three years. It is anticipated that genetically modified organisms (GMOs) for food purposes will be widely produced using the clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR)/Cas9 system in the near future. However, the Cas9 gene may then enter the genomes of GMOs for food if the breeding process is not strictly managed, which could lead to the Cas9 protein or associated peptides being produced within these organisms. A variety of peptides could theoretically be produced from the Cas9 gene by using open reading frames different from that of Cas9 in the GMOs. In this study, Cas9 and the peptides potentially encoded by Cas9 genes were studied regarding their immunogenicity, in terms of the digestibility of Cas9 and the homology of the peptides to food allergens. First, the digestibility and thermal stability of Cas9 were studied. Digestibility was tested with natural or heat-denatured Cas9 in simulated gastric fluid in vitro. The two types of Cas9 were digested rapidly. Cas9 was also gradually degraded during heat treatment. Second, the peptides potentially encoded by Cas9 genes were examined for their homology to food allergens. Specifically, an 8-mer exact match search and a sliding 80-mer window search were performed using allergen databases. One of the peptides was found to have homology with a food allergen.

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confidence: 99%

mentioning

confidence: 99%

Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy

Nakajima

Nishimaki-Mogami

Kondo

2016

Biological & Pharmaceutical Bulletin

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“…There are multiple groups that have used these tools to produce myostatin knockout animals, including cattle (Proudfoot et al 2015), goats (Ni et al 2014), and fish . These knockout animals are expected to have similar muscle hypertrophy phenotypes to those breeds with naturally occurring myostatin mutations.…”

Section: Altering Developmental Programsmentioning

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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“…Designer animals are not far behind. Scientists in China have successfully deleted two genes from earlystage goat embryos that suppress both hair and muscle growth, giving birth to kids exhibiting both larger muscles and longer fur, paving the way for creation of designer animals to be used for research 13 . Wang et al…”

Section: Redesigning Nature: Use Of Cas/crispr In Genome Editingmentioning

confidence: 99%