Generation of a precise Oct4-hrGFP knockin cynomolgus monkey model via CRISPR/Cas9-assisted homologous recombination

Cui, Yiqiang; Niu, Yuyu; Zhou, Jianghua; Chen, Yongchang; Cheng, Yiwei; Li, Shangang; Ai, Zongyong; Chu, Chu; Wang, Hong; Zheng, Bo; Chen, Xuejin; Sha, Jiahao; Guo, Xuejiang; Huang, Xingxu; Ji, Weizhi

doi:10.1038/cr.2018.10

Cited by 44 publications

(40 citation statements)

References 7 publications

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“…1c, 1d, Table 1), concordant with the pattern seen in rhesus monkeys. For the sgRNA-predicted off-target sites, only one mutation (a 2-bp deletion) was seen in the knockin cynomolgus monkey as reported in Cai et al 19 .…”

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confidence: 68%

“…To evaluate the situation in monkeys, we performed trio WGS of Cas9-edited rhesus monkeys (Macaca mulatta). We also analyzed the published trio WGS data of Cas9-edited cynomolgus monkeys (Macaca fascicularis) 19 .…”

mentioning

confidence: 99%

“…To further confirm the DNM pattern seen in rhesus monkeys, using the same pipeline, we analyzed the published trio WGS data of a gene-knockin model via CRISPR-Cas9 in cynomolgus monkeys 19 . The trio included an Oct4-hrGFP knockin cynomolgus monkey and his two wildtype parents ( Fig.…”

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confidence: 99%

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Trio deep-sequencing does not reveal unexpected mutations in Cas9-edited monkeys

Luo

Zhang³

et al. 2018

Preprint

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CRISPR-Cas9 is a widely-used genome editing tool, but its off-target effect remains a concern, especially in view of future clinical applications. Non-human primates (NHPs) share close genetic and physiological similarities with humans, making them an ideal preclinical model for developing Cas9-based therapies. However, no comprehensive in vivo off-target assessment has been conducted in NHPs. Here we performed whole genome trio sequencing of Cas9-treated monkeys. We found they only carried a small number of de novo mutations that can be explained by expected spontaneous mutations, and no unexpected mutations were detected.

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confidence: 68%

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confidence: 99%

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Trio deep-sequencing does not reveal unexpected mutations in Cas9-edited monkeys

Luo

Zhang³

et al. 2018

Preprint

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“…The relative merits of these have been extensively reviewed elsewhere (Van Otterloo, Williams, & Artinger, 2016). Larger animal models can also be created including goats, pigs, and monkeys with CRISPR-Cas9 editing (Cui et al, 2018;Hai, Teng, Guo, Li, & Zhou, 2014;Ni et al, 2014). We favor the mouse models for a combination of accessible genetics, relative cost, fast generation time, and physical resemblance to human biology as compared to many other genetic models.…”

Section: Discussionmentioning

confidence: 99%

Using human sequencing to guide craniofacial research

Liegel

Finnerty

Blizzard

et al. 2018

Genesis

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Summary A recent convergence of technological innovations has re‐energized the ability to apply genetics to research in human craniofacial development. Next‐generation exome and whole genome sequencing have significantly dropped in price, making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR‐Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here, we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggested a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.

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“…The relative merits of these have been extensively reviewed elsewhere, e.g., (Van Otterloo, Williams, & Artinger, 2016). Larger animal models can also be created including goats, pigs and monkeys with CRISPR-Cas9 editing (Cui et al, 2018; Hai, Teng, Guo, Li, & Zhou, 2014; Ni et al, 2014). We favor the mouse models for a combination of accessible genetics, relative cost, fast generation time, and physical resemblance to human biology as compared to many other genetic models.…”

Section: Introductionmentioning

confidence: 99%

Using human sequencing to guide craniofacial research

Liegel

Finnerty

Ward

et al. 2018

Preprint

View full text Add to dashboard Cite

A recent convergence of technological innovations has re-energized the ability to apply genetics to research in human craniofacial development. Next-generation exome and whole genome sequencing have dropped in price significantly making it relatively trivial to sequence and analyze patients and families with congenital craniofacial anomalies. A concurrent revolution in genome editing with the use of the CRISPR-Cas9 system enables the rapid generation of animal models, including mouse, which can precisely recapitulate human variants. Here we summarize the choices currently available to the research community. We illustrate this approach with the study of a family with a novel craniofacial syndrome with dominant inheritance pattern. The genomic analysis suggest a causal variant in AMOTL1 which we modeled in mice. We also made a novel deletion allele of Amotl1. Our results indicate that Amotl1 is not required in the mouse for survival to weaning. Mice carrying the variant identified in the human sequencing studies, however, do not survive to weaning in normal ratios. The cause of death is not understood for these mice complicating our conclusions about the pathogenicity in the index patient. Thus, we highlight some of the powerful opportunities and confounding factors confronting current craniofacial genetic research.

show abstract

Generation of a precise Oct4-hrGFP knockin cynomolgus monkey model via CRISPR/Cas9-assisted homologous recombination

Cited by 44 publications

References 7 publications

Trio deep-sequencing does not reveal unexpected mutations in Cas9-edited monkeys

Trio deep-sequencing does not reveal unexpected mutations in Cas9-edited monkeys

Using human sequencing to guide craniofacial research

Using human sequencing to guide craniofacial research

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