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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...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Korean Society of Veterinary Science
2019
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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. |
format | Online Article Text |
id | pubmed-6538514 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | The Korean Society of Veterinary Science |
record_format | MEDLINE/PubMed |
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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