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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...

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Autores principales: Chen, Yi-Jiun, Cheng, Ya-Yun, Wang, Weikang, Tian, Xiao-Jun, Lefever, Daniel E, Taft, David A, Zhang, Jingyu, Xing, Jianhua
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
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.
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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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