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Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in
Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing techniques find applications in many fields, such as molecular biology, cancer biology, and disease modeling. In contrast to the knock-out procedure, a key step of CRISPR knock-in experiments is the homology-direct...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211398/ https://www.ncbi.nlm.nih.gov/pubmed/32411820 http://dx.doi.org/10.1093/biomethods/bpaa006 |
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author | Chen, Yi-Jiun Cheng, Ya-Yun Wang, Weikang Tian, Xiao-Jun Lefever, Daniel E Taft, David A Zhang, Jingyu Xing, Jianhua |
author_facet | Chen, Yi-Jiun Cheng, Ya-Yun Wang, Weikang Tian, Xiao-Jun Lefever, Daniel E Taft, David A Zhang, Jingyu Xing, Jianhua |
author_sort | Chen, Yi-Jiun |
collection | PubMed |
description | Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing techniques find applications in many fields, such as molecular biology, cancer biology, and disease modeling. In contrast to the knock-out procedure, a key step of CRISPR knock-in experiments is the homology-directed repair process that requires donor constructs as repair templates. Therefore, it is desirable to generate a series of donor templates efficiently and cost-effectively. In this study, we developed a new strategy that combines (i) Gibson assembly reaction, (ii) a linker pair composed of eight in silico screened restriction enzyme sites, and (iii) a hierarchical framework, to remarkably improve the efficiency of producing donor constructs for common genes as well as for the genes containing unbalanced guanine-cytosine content and requiring a selectable marker. Furthermore, the approach provides the ability of inserting additional elements into the donor templates, such as single guide RNA recognition sites that have been reported to enhance the efficiency of homology-directed repair. Conclusively, our modularized process is simple, fast, and cost-effective for making donor constructs and benefits the application of CRISPR knock-in methods. |
format | Online Article Text |
id | pubmed-7211398 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-72113982020-05-14 Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in Chen, Yi-Jiun Cheng, Ya-Yun Wang, Weikang Tian, Xiao-Jun Lefever, Daniel E Taft, David A Zhang, Jingyu Xing, Jianhua Biol Methods Protoc Methods Manuscript Clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing techniques find applications in many fields, such as molecular biology, cancer biology, and disease modeling. In contrast to the knock-out procedure, a key step of CRISPR knock-in experiments is the homology-directed repair process that requires donor constructs as repair templates. Therefore, it is desirable to generate a series of donor templates efficiently and cost-effectively. In this study, we developed a new strategy that combines (i) Gibson assembly reaction, (ii) a linker pair composed of eight in silico screened restriction enzyme sites, and (iii) a hierarchical framework, to remarkably improve the efficiency of producing donor constructs for common genes as well as for the genes containing unbalanced guanine-cytosine content and requiring a selectable marker. Furthermore, the approach provides the ability of inserting additional elements into the donor templates, such as single guide RNA recognition sites that have been reported to enhance the efficiency of homology-directed repair. Conclusively, our modularized process is simple, fast, and cost-effective for making donor constructs and benefits the application of CRISPR knock-in methods. Oxford University Press 2020-03-20 /pmc/articles/PMC7211398/ /pubmed/32411820 http://dx.doi.org/10.1093/biomethods/bpaa006 Text en © The Author(s) 2020. Published by Oxford University Press. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Manuscript Chen, Yi-Jiun Cheng, Ya-Yun Wang, Weikang Tian, Xiao-Jun Lefever, Daniel E Taft, David A Zhang, Jingyu Xing, Jianhua Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in |
title | Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in |
title_full | Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in |
title_fullStr | Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in |
title_full_unstemmed | Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in |
title_short | Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in |
title_sort | rapid, modular, and cost-effective generation of donor dna constructs for crispr-based gene knock-in |
topic | Methods Manuscript |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7211398/ https://www.ncbi.nlm.nih.gov/pubmed/32411820 http://dx.doi.org/10.1093/biomethods/bpaa006 |
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