CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs

Wang, Kankan; Tang, Xiaochun; Xie, Zhixun; Zou, Xiaodong; Li, Mengjing; Yuan, Hongming; Guo, Ning; Ouyang, Hongsheng; Jiao, Huping; Pang, Daxin

doi:10.1007/s11248-017-0044-z

Cited by 76 publications

(43 citation statements)

References 24 publications

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“…In contrast, the introduction of a single point mutation using designer nuclease-based genome editing is very inefficient, and the efficacy depends on the respective cell type [ 22 ]. Porcine fetal fibroblasts have been used successfully for genome editing followed by SCNT-based cloning, both for knock-outs [ 23 , 24 , 25 ] and also for knock-ins [ 21 ]. Nevertheless, total knock-in efficacy remains low in these cells, for an efficient production of genome edited pigs via SCNT, this ratio needs to be improved.…”

Section: Discussionmentioning

confidence: 99%

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene

Gerlach

Kraft

Brenner

et al. 2018

Genes

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During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7-gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (GMNN). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9-GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9-GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.

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Section: Discussionmentioning

confidence: 99%

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene

Gerlach

Kraft

Brenner

et al. 2018

Genes

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“…MSTN modification is an effective approach to enhance muscle growth in various animals. Given that this gene is a “hot” candidate that is possibly beneficial in agriculture, several strains of MSTN-knockout pigs have been generated by using ZFNs ( Qian et al, 2015 ), TALENs ( Rao et al, 2016 ), and CRISPR/Cas9 ( Wang et al, 2015 , 2017b ; Bi et al, 2016 ). These MSTN-knockout pigs demonstrate muscle hypertrophy or double-muscled (DM) phenotype, with increased muscle mass and decreased fat accumulation compared with wild-type (WT) pigs.…”

Section: Genetically Modified Pigs For Agricultural Applicationmentioning

confidence: 99%

“…With the improved efficiency of genetic modification, pig genome modification can confer any desired, predetermined genetic changes, which would take years to be realized in traditional selective breeding. Numerous economically significant characteristics, such as increased meat production ( Qian et al, 2015 ; Wang et al, 2015 , 2017b ; Bi et al, 2016 ; Rao et al, 2016 ), reduced fat deposition ( Zheng et al, 2017 ), or enhanced disease resistance ( Whitworth et al, 2016 ; Burkard et al, 2017 ; Wells et al, 2017 ; Yang et al, 2018 ), have been achieved simply and efficiently through genetic modification in pigs, which can be used as valuable breeding materials to advance pig production. In biomedical research, pigs serve as an important large animal model given their advantages over other models.…”

Section: Introductionmentioning

confidence: 99%

Genome Editing of Pigs for Agriculture and Biomedicine

Yang

2018

Front. Genet.

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Pigs serve as an important agricultural resource and animal model in biomedical studies. Efficient and precise modification of pig genome by using recently developed gene editing tools has significantly broadened the application of pig models in various research areas. The three types of site-specific nucleases, namely, zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein, are the main gene editing tools that can efficiently introduce predetermined modifications, including knockouts and knockins, into the pig genome. These modifications can confer desired phenotypes to pigs to improve production traits, such as optimal meat production, enhanced feed digestibility, and disease resistance. Besides, given their genetic, anatomic, and physiologic similarities to humans, pigs can also be modified to model human diseases or to serve as an organ source for xenotransplantation to save human lives. To date, many genetically modified pig models with agricultural or biomedical values have been established by using gene editing tools. These pig models are expected to accelerate research progress in related fields and benefit humans.

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“…More recently, the CRISPR/Cas9 system was employed to genetically manipulate pigs, rabbits, and goats to disable the myostatin gene with the goal of increasing meat production (Guo et al, 2016;Wang et al, 2017). In all species, the resulting animals were significantly more muscular.…”

Section: Myostatinmentioning

confidence: 99%

Invited review: Genetic and genomic mouse models for livestock research

2018

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Abstract. Knowledge about the function and functioning of single or multiple interacting genes is of the utmost significance for understanding the organism as a whole and for accurate livestock improvement through genomic selection. This includes, but is not limited to, understanding the ontogenetic and environmentally driven regulation of gene action contributing to simple and complex traits. Genetically modified mice, in which the functions of single genes are annotated; mice with reduced genetic complexity; and simplified structured populations are tools to gain fundamental knowledge of inheritance patterns and whole system genetics and genomics. In this review, we briefly describe existing mouse resources and discuss their value for fundamental and applied research in livestock.

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CRISPR/Cas9-mediated knockout of myostatin in Chinese indigenous Erhualian pigs

Cited by 76 publications

References 24 publications

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene

Genome Editing of Pigs for Agriculture and Biomedicine

Invited review: Genetic and genomic mouse models for livestock research

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