CRISPR/Cas9-mediated genome editing in nonhuman primates

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ABSTRACT

Owing to their high similarity to humans, non-human primates (NHPs) provide an exceedingly suitable model for the study of human disease. In this Review, we summarize the history of transgenic NHP models and the progress of CRISPR/Cas9-mediated genome editing in NHPs, from the first proof-of-principle green fluorescent protein-expressing monkeys to sophisticated NHP models of human neurodegenerative disease that accurately phenocopy several complex disease features. We discuss not only the breakthroughs and advantages, but also the potential shortcomings of the application of the CRISPR/Cas9 system to NHPs that have emerged from the expanded understanding of this technology in recent years. Although off-target and mosaic mutations are the main concerns in CRISPR/Cas9-mediated NHP modeling, recent progress in genome editing techniques make it likely that these technical limitations will be overcome soon, bringing excellent prospects to human disease studies.

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Summary: This Review discusses the history and development of genome editing in non-human primates, as well as the challenges and prospects facing this technology now and in the future.

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Most cited references 30

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Adenoviruses as vaccine vectors

Nia Tatsis, Hildegund Ertl (2004)

Adenoviruses have transitioned from tools for gene replacement therapy to bona fide vaccine delivery vehicles. They are attractive vaccine vectors as they induce both innate and adaptive immune responses in mammalian hosts. Currently, adenovirus vectors are being tested as subunit vaccine systems for numerous infectious agents ranging from malaria to HIV-1. Additionally, they are being explored as vaccines against a multitude of tumor-associated antigens. In this review we describe the molecular biology of adenoviruses as well as ways the adenovirus vectors can be manipulated to enhance their efficacy as vaccine carriers. We describe methods of evaluating immune responses to transgene products expressed by adenoviral vectors and discuss data on adenoviral vaccines to a selected number of pathogens. Last, we comment on the limitations of using human adenoviral vectors and provide alternatives to circumvent these problems. This field is growing at an exciting and rapid pace, thus we have limited our scope to the use of adenoviral vectors as vaccines against viral pathogens.

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Towards a transgenic model of Huntington's disease in a non-human primate.

Shang-Hsun Yang, Pei-Hsun Cheng, Heather Banta … (2008)

Non-human primates are valuable for modelling human disorders and for developing therapeutic strategies; however, little work has been reported in establishing transgenic non-human primate models of human diseases. Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive deterioration and psychiatric disturbances followed by death within 10-15 years of the onset of the symptoms. HD is caused by the expansion of cytosine-adenine-guanine (CAG, translated into glutamine) trinucleotide repeats in the first exon of the human huntingtin (HTT) gene. Mutant HTT with expanded polyglutamine (polyQ) is widely expressed in the brain and peripheral tissues, but causes selective neurodegeneration that is most prominent in the striatum and cortex of the brain. Although rodent models of HD have been developed, these models do not satisfactorily parallel the brain changes and behavioural features observed in HD patients. Because of the close physiological, neurological and genetic similarities between humans and higher primates, monkeys can serve as very useful models for understanding human physiology and diseases. Here we report our progress in developing a transgenic model of HD in a rhesus macaque that expresses polyglutamine-expanded HTT. Hallmark features of HD, including nuclear inclusions and neuropil aggregates, were observed in the brains of the HD transgenic monkeys. Additionally, the transgenic monkeys showed important clinical features of HD, including dystonia and chorea. A transgenic HD monkey model may open the way to understanding the underlying biology of HD better, and to the development of potential therapies. Moreover, our data suggest that it will be feasible to generate valuable non-human primate models of HD and possibly other human genetic diseases.

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A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture

Henriette O'Geen, Isabelle M. Henry, Mital S. Bhakta … (2015)

Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided nucleases have gathered considerable excitement as a tool for genome engineering. However, questions remain about the specificity of target site recognition. Cleavage specificity is typically evaluated by low throughput assays (T7 endonuclease I assay, target amplification followed by high-throughput sequencing), which are limited to a subset of potential off-target sites. Here, we used ChIP-seq to examine genome-wide CRISPR binding specificity at gRNA-specific and gRNA-independent sites for two guide RNAs. RNA-guided Cas9 binding was highly specific to the target site while off-target binding occurred at much lower intensities. Cas9-bound regions were highly enriched in NGG sites, a sequence required for target site recognition by Streptococcus pyogenes Cas9. To determine the relationship between Cas9 binding and endonuclease activity, we applied targeted sequence capture, which allowed us to survey 1200 genomic loci simultaneously including potential off-target sites identified by ChIP-seq and by computational prediction. A high frequency of indels was observed at both target sites and one off-target site, while no cleavage activity could be detected at other ChIP-bound regions. Our results confirm the high-specificity of CRISPR endonucleases and demonstrate that sequence capture can be used as a high-throughput genome-wide approach to identify off-target activity.

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Journal

Journal ID (nlm-ta): Dis Model Mech

Journal ID (iso-abbrev): Dis Model Mech

Journal ID (publisher-id): DMM

Journal ID (hwp): dmm

Title: Disease Models & Mechanisms

Publisher: The Company of Biologists Ltd

ISSN (Print): 1754-8403

ISSN (Electronic): 1754-8411

Publication date (Print, pub): 1 October 2019

Publication date (Electronic): 16 October 2019

Publication date PMC-release: 16 October 2019

Volume: 12

Issue: 10

Electronic Location Identifier: dmm039982

Affiliations

[1 ]Faculty of Environmental Science and Engineering, Kunming University of Science and Technology , Kunming, Yunnan 650500, China

[2 ]Institute of Primate Translational Medicine, Kunming University of Science and Technology , Kunming, Yunnan 650500, China

[3 ]Yunnan Key Laboratory of Primate Biomedicine Research , Kunming, Yunnan 650223, China

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[*]

These authors contributed equally to this work

[‡ ]Author for correspondence ( Niuyy@ 123456lpbr.cn )

Author information

Yu Kang http://orcid.org/0000-0003-1730-6498

Chu Chu http://orcid.org/0000-0002-8680-6149

Fang Wang http://orcid.org/0000-0001-8220-5226

Yuyu Niu http://orcid.org/0000-0002-8797-4152

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Publisher ID: DMM039982

DOI: 10.1242/dmm.039982

PMC ID: 6826017

PubMed ID: 31636095

SO-VID: 23deaa74-bd20-4933-9b84-0aef777308ab

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This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Funded by: National Key Research and Development Program of China, Ministry of Science and Technology of the People's Republic of China, http://dx.doi.org/10.13039/501100002855;

CRISPR/Cas9-mediated genome editing in nonhuman primates

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CRISPR/Cas9 editing in human blood

Most cited references 30

Adenoviruses as vaccine vectors

Towards a transgenic model of Huntington's disease in a non-human primate.

A genome-wide analysis of Cas9 binding specificity using ChIP-seq and targeted sequence capture

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