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sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9
The SNP within intron 3 of the porcine IGF2 gene (G3072A) plays an important role for muscle growth and fat deposition in pigs. In this study, the StCas9 derived from Streptococcus thermophilus together with the Drosha-mediated sgRNA-shRNA structure were combined to boost the G to A base editing on...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482158/ https://www.ncbi.nlm.nih.gov/pubmed/31057603 http://dx.doi.org/10.3389/fgene.2019.00347 |
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author | Sun, Yongsen Yan, Nana Mu, Lu Sun, Bing Deng, Jingrong Fang, Yuanyuan Shao, Simin Yan, Qiang Han, Furong Zhang, Zhiying Xu, Kun |
author_facet | Sun, Yongsen Yan, Nana Mu, Lu Sun, Bing Deng, Jingrong Fang, Yuanyuan Shao, Simin Yan, Qiang Han, Furong Zhang, Zhiying Xu, Kun |
author_sort | Sun, Yongsen |
collection | PubMed |
description | The SNP within intron 3 of the porcine IGF2 gene (G3072A) plays an important role for muscle growth and fat deposition in pigs. In this study, the StCas9 derived from Streptococcus thermophilus together with the Drosha-mediated sgRNA-shRNA structure were combined to boost the G to A base editing on the IGF2 SNP site, which we called “SNP editing.” The codon-humanized StCas9 as we previously reported was firstly compared with the prevalently used SpCas9 derived from Streptococcus pyogenes using our idiomatic surrogate report assay, and the StCas9 demonstrated a comparable targeting activity. On the other hand, by combining shRNA with sgRNA, simultaneous gene silencing and genome targeting can be achieved. Thus, the novel IGF2.sgRNA-LIG4.shRNA-IGF2.sgRNA structure was constructed to enhance the sgRNA/Cas9-mediated HDR-based IGF2 SNP editing by silencing the LIG4 gene, which is a key molecule of the HDR’s competitive NHEJ pathway. The sgRNA-shRNA/StCas9 all-in-one expression vector and the IGF2.sgRNA/StCas9 as control were separately used to transfect porcine PK15 cells together with an ssODNs donor for the IGF2 SNP editing. The editing events were detected by the RFLP assay, Sanger sequencing as well as Deep-sequencing, and the Deep-sequencing results finally demonstrated a significant higher HDR-based editing efficiency (16.38%) for our sgRNA-shRNA/StCas9 strategy. In short, we achieved effective IGF2 SNP editing by using the combined sgRNA-shRNA/StCas9 strategy, which will facilitate the further production of base-edited animals and perhaps extend for the gene therapy for the base correction of some genetic diseases. |
format | Online Article Text |
id | pubmed-6482158 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-64821582019-05-03 sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9 Sun, Yongsen Yan, Nana Mu, Lu Sun, Bing Deng, Jingrong Fang, Yuanyuan Shao, Simin Yan, Qiang Han, Furong Zhang, Zhiying Xu, Kun Front Genet Genetics The SNP within intron 3 of the porcine IGF2 gene (G3072A) plays an important role for muscle growth and fat deposition in pigs. In this study, the StCas9 derived from Streptococcus thermophilus together with the Drosha-mediated sgRNA-shRNA structure were combined to boost the G to A base editing on the IGF2 SNP site, which we called “SNP editing.” The codon-humanized StCas9 as we previously reported was firstly compared with the prevalently used SpCas9 derived from Streptococcus pyogenes using our idiomatic surrogate report assay, and the StCas9 demonstrated a comparable targeting activity. On the other hand, by combining shRNA with sgRNA, simultaneous gene silencing and genome targeting can be achieved. Thus, the novel IGF2.sgRNA-LIG4.shRNA-IGF2.sgRNA structure was constructed to enhance the sgRNA/Cas9-mediated HDR-based IGF2 SNP editing by silencing the LIG4 gene, which is a key molecule of the HDR’s competitive NHEJ pathway. The sgRNA-shRNA/StCas9 all-in-one expression vector and the IGF2.sgRNA/StCas9 as control were separately used to transfect porcine PK15 cells together with an ssODNs donor for the IGF2 SNP editing. The editing events were detected by the RFLP assay, Sanger sequencing as well as Deep-sequencing, and the Deep-sequencing results finally demonstrated a significant higher HDR-based editing efficiency (16.38%) for our sgRNA-shRNA/StCas9 strategy. In short, we achieved effective IGF2 SNP editing by using the combined sgRNA-shRNA/StCas9 strategy, which will facilitate the further production of base-edited animals and perhaps extend for the gene therapy for the base correction of some genetic diseases. Frontiers Media S.A. 2019-04-18 /pmc/articles/PMC6482158/ /pubmed/31057603 http://dx.doi.org/10.3389/fgene.2019.00347 Text en Copyright © 2019 Sun, Yan, Mu, Sun, Deng, Fang, Shao, Yan, Han, Zhang and Xu. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Genetics Sun, Yongsen Yan, Nana Mu, Lu Sun, Bing Deng, Jingrong Fang, Yuanyuan Shao, Simin Yan, Qiang Han, Furong Zhang, Zhiying Xu, Kun sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9 |
title | sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9 |
title_full | sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9 |
title_fullStr | sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9 |
title_full_unstemmed | sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9 |
title_short | sgRNA-shRNA Structure Mediated SNP Site Editing on Porcine IGF2 Gene by CRISPR/StCas9 |
title_sort | sgrna-shrna structure mediated snp site editing on porcine igf2 gene by crispr/stcas9 |
topic | Genetics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482158/ https://www.ncbi.nlm.nih.gov/pubmed/31057603 http://dx.doi.org/10.3389/fgene.2019.00347 |
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