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A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection
Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer (SCNT). However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Science China Press
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8817169/ https://www.ncbi.nlm.nih.gov/pubmed/35122622 http://dx.doi.org/10.1007/s11427-021-2058-2 |
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author | Xu, Kui Zhang, Xiuling Liu, Zhiguo Ruan, Jinxue Xu, Changjiang Che, Jingjing Fan, Ziyao Mu, Yulian Li, Kui |
author_facet | Xu, Kui Zhang, Xiuling Liu, Zhiguo Ruan, Jinxue Xu, Changjiang Che, Jingjing Fan, Ziyao Mu, Yulian Li, Kui |
author_sort | Xu, Kui |
collection | PubMed |
description | Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer (SCNT). However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP (reporter RNA enriched dual-sgRNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sgRNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs (CRISPR-Cas9 ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3–4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1 (WIP1) gene: among 32 weaned cloned pigs, 31 (97%) carried WIP1 edits, and 15 (47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout (KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. RE-DSRNP’s strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals. SUPPORTING INFORMATION: The supporting information is available online at 10.1007/s11427-021-2058-2. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors. |
format | Online Article Text |
id | pubmed-8817169 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Science China Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-88171692022-02-07 A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection Xu, Kui Zhang, Xiuling Liu, Zhiguo Ruan, Jinxue Xu, Changjiang Che, Jingjing Fan, Ziyao Mu, Yulian Li, Kui Sci China Life Sci Research Paper Gene-edited pigs for agricultural and biomedical applications are typically generated using somatic cell nuclear transfer (SCNT). However, SCNT requires the use of monoclonal cells as donors, and the time-consuming and laborious monoclonal selection process limits the production of large populations of gene-edited animals. Here, we developed a rapid and efficient method named RE-DSRNP (reporter RNA enriched dual-sgRNA/CRISPR-Cas9 ribonucleoproteins) for generating gene-edited donor cells. RE-DSRNP takes advantage of the precise and efficient editing features of dual-sgRNA and the high editing efficiency, low off-target effects, transgene-free nature, and low cytotoxic characteristics of reporter RNA enriched RNPs (CRISPR-Cas9 ribonucleoproteins), thus eliminating the need for the selection of monoclonal cells and thereby greatly reducing the generation time of donor cells from 3–4 weeks to 1 week, while also reducing the extent of apoptosis and chromosomal aneuploidy of donor cells. We applied RE-DSRNP to produce cloned pigs bearing a deletion edit of the wild-type p53-induced phosphatase 1 (WIP1) gene: among 32 weaned cloned pigs, 31 (97%) carried WIP1 edits, and 15 (47%) were homozygous for the designed fragment deletion, and no off-target event was detected. The WIP1 knockout (KO) pigs exhibited male reproductive disorders, illustrating the utility of RE-DSRNP for rapidly generating precisely edited animals for functional genomics and disease research. RE-DSRNP’s strong editing performance in a large animal and its marked reduction in the required time for producing SCNT donor cells support its application prospects for rapidly generating populations of transgene-free cloned animals. SUPPORTING INFORMATION: The supporting information is available online at 10.1007/s11427-021-2058-2. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors. Science China Press 2022-01-28 2022 /pmc/articles/PMC8817169/ /pubmed/35122622 http://dx.doi.org/10.1007/s11427-021-2058-2 Text en © Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2022 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. |
spellingShingle | Research Paper Xu, Kui Zhang, Xiuling Liu, Zhiguo Ruan, Jinxue Xu, Changjiang Che, Jingjing Fan, Ziyao Mu, Yulian Li, Kui A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection |
title | A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection |
title_full | A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection |
title_fullStr | A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection |
title_full_unstemmed | A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection |
title_short | A transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection |
title_sort | transgene-free method for rapid and efficient generation of precisely edited pigs without monoclonal selection |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8817169/ https://www.ncbi.nlm.nih.gov/pubmed/35122622 http://dx.doi.org/10.1007/s11427-021-2058-2 |
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