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The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the lengt...

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Autores principales: Lv, Jiawei, Wu, Shuang, Wei, Renyue, Li, Yan, Jin, Junxue, Mu, Yanshuang, Zhang, Yu, Kong, Qingran, Weng, Xiaogang, Liu, Zhonghua
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Korean Society of Veterinary Science 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538514/
https://www.ncbi.nlm.nih.gov/pubmed/31161741
http://dx.doi.org/10.4142/jvs.2019.20.e23
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author Lv, Jiawei
Wu, Shuang
Wei, Renyue
Li, Yan
Jin, Junxue
Mu, Yanshuang
Zhang, Yu
Kong, Qingran
Weng, Xiaogang
Liu, Zhonghua
author_facet Lv, Jiawei
Wu, Shuang
Wei, Renyue
Li, Yan
Jin, Junxue
Mu, Yanshuang
Zhang, Yu
Kong, Qingran
Weng, Xiaogang
Liu, Zhonghua
author_sort Lv, Jiawei
collection PubMed
description The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.
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spelling pubmed-65385142019-06-04 The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells Lv, Jiawei Wu, Shuang Wei, Renyue Li, Yan Jin, Junxue Mu, Yanshuang Zhang, Yu Kong, Qingran Weng, Xiaogang Liu, Zhonghua J Vet Sci Original Article The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells. The Korean Society of Veterinary Science 2019-05 2019-05-03 /pmc/articles/PMC6538514/ /pubmed/31161741 http://dx.doi.org/10.4142/jvs.2019.20.e23 Text en © 2019 The Korean Society of Veterinary Science https://creativecommons.org/licenses/by-nc/4.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Article
Lv, Jiawei
Wu, Shuang
Wei, Renyue
Li, Yan
Jin, Junxue
Mu, Yanshuang
Zhang, Yu
Kong, Qingran
Weng, Xiaogang
Liu, Zhonghua
The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells
title The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells
title_full The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells
title_fullStr The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells
title_full_unstemmed The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells
title_short The length of guide RNA and target DNA heteroduplex effects on CRISPR/Cas9 mediated genome editing efficiency in porcine cells
title_sort length of guide rna and target dna heteroduplex effects on crispr/cas9 mediated genome editing efficiency in porcine cells
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538514/
https://www.ncbi.nlm.nih.gov/pubmed/31161741
http://dx.doi.org/10.4142/jvs.2019.20.e23
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