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Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor
Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 ba...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2021
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928300/ https://www.ncbi.nlm.nih.gov/pubmed/33681181 http://dx.doi.org/10.3389/fcell.2020.598634 |
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author | Ranawakage, Deshani C. Okada, Keita Sugio, Kota Kawaguchi, Yuya Kuninobu-Bonkohara, Yuki Takada, Takuya Kamachi, Yusuke |
author_facet | Ranawakage, Deshani C. Okada, Keita Sugio, Kota Kawaguchi, Yuya Kuninobu-Bonkohara, Yuki Takada, Takuya Kamachi, Yusuke |
author_sort | Ranawakage, Deshani C. |
collection | PubMed |
description | Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently “knocked-in” into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3′ homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5′ and 3′ junctions indicated that knock-in allele frequencies were higher at the 3′ side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos. |
format | Online Article Text |
id | pubmed-7928300 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-79283002021-03-04 Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor Ranawakage, Deshani C. Okada, Keita Sugio, Kota Kawaguchi, Yuya Kuninobu-Bonkohara, Yuki Takada, Takuya Kamachi, Yusuke Front Cell Dev Biol Cell and Developmental Biology Despite the unprecedented gene editing capability of CRISPR-Cas9-mediated targeted knock-in, the efficiency and precision of this technology still require further optimization, particularly for multicellular model organisms, such as the zebrafish (Danio rerio). Our study demonstrated that an ∼200 base-pair sequence encoding a composite tag can be efficiently “knocked-in” into the zebrafish genome using a combination of the CRISPR-Cas9 ribonucleoprotein complex and a long single-stranded DNA (lssDNA) as a donor template. Here, we targeted the sox3, sox11a, and pax6a genes to evaluate the knock-in efficiency of lssDNA donors with different structures in somatic cells of injected embryos and for their germline transmission. The structures and sequence characteristics of the lssDNA donor templates were found to be crucial to achieve a high rate of precise and heritable knock-ins. The following were our key findings: (1) lssDNA donor strand selection is important; however, strand preference and its dependency appear to vary among the target loci or their sequences. (2) The length of the 3′ homology arm of the lssDNA donor affects knock-in efficiency in a site-specific manner; particularly, a shorter 50-nt arm length leads to a higher knock-in efficiency than a longer 300-nt arm for the sox3 and pax6a knock-ins. (3) Some DNA sequence characteristics of the knock-in donors and the distance between the CRISPR-Cas9 cleavage site and the tag insertion site appear to adversely affect the repair process, resulting in imprecise editing. By implementing the proposed method, we successfully obtained precisely edited sox3, sox11a, and pax6a knock-in alleles that contained a composite tag composed of FLAGx3 (or PAx3), Bio tag, and HiBiT tag (or His tag) with moderate to high germline transmission rates as high as 21%. Furthermore, the knock-in allele-specific quantitative polymerase chain reaction (qPCR) for both the 5′ and 3′ junctions indicated that knock-in allele frequencies were higher at the 3′ side of the lssDNAs, suggesting that the lssDNA-templated knock-in was mediated by unidirectional single-strand template repair (SSTR) in zebrafish embryos. Frontiers Media S.A. 2021-02-11 /pmc/articles/PMC7928300/ /pubmed/33681181 http://dx.doi.org/10.3389/fcell.2020.598634 Text en Copyright © 2021 Ranawakage, Okada, Sugio, Kawaguchi, Kuninobu-Bonkohara, Takada and Kamachi. 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 | Cell and Developmental Biology Ranawakage, Deshani C. Okada, Keita Sugio, Kota Kawaguchi, Yuya Kuninobu-Bonkohara, Yuki Takada, Takuya Kamachi, Yusuke Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor |
title | Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor |
title_full | Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor |
title_fullStr | Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor |
title_full_unstemmed | Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor |
title_short | Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor |
title_sort | efficient crispr-cas9-mediated knock-in of composite tags in zebrafish using long ssdna as a donor |
topic | Cell and Developmental Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928300/ https://www.ncbi.nlm.nih.gov/pubmed/33681181 http://dx.doi.org/10.3389/fcell.2020.598634 |
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