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...
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Frontiers Media S.A.
2021-02-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcell.2020.598634/full |
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| author | Deshani C. Ranawakage Keita Okada Kota Sugio Yuya Kawaguchi Yuki Kuninobu-Bonkohara Takuya Takada Yusuke Kamachi |
| author_facet | Deshani C. Ranawakage Keita Okada Kota Sugio Yuya Kawaguchi Yuki Kuninobu-Bonkohara Takuya Takada Yusuke Kamachi |
| author_sort | Deshani C. Ranawakage |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T12:10:20Z |
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| institution | Directory Open Access Journal |
| issn | 2296-634X |
| language | English |
| last_indexed | 2024-12-14T12:10:20Z |
| publishDate | 2021-02-01 |
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| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj.art-0c8dc2dc68eb4a5ebc6159214f78efde2022-12-21T23:01:45ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2021-02-01810.3389/fcell.2020.598634598634Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a DonorDeshani C. RanawakageKeita OkadaKota SugioYuya KawaguchiYuki Kuninobu-BonkoharaTakuya TakadaYusuke KamachiDespite 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.https://www.frontiersin.org/articles/10.3389/fcell.2020.598634/fullCRISPR-Cas9long ssDNA donorknock-inendogenous taggingSoxB1composite tag |
| spellingShingle | Deshani C. Ranawakage Keita Okada Kota Sugio Yuya Kawaguchi Yuki Kuninobu-Bonkohara Takuya Takada Yusuke Kamachi Efficient CRISPR-Cas9-Mediated Knock-In of Composite Tags in Zebrafish Using Long ssDNA as a Donor Frontiers in Cell and Developmental Biology CRISPR-Cas9 long ssDNA donor knock-in endogenous tagging SoxB1 composite tag |
| 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 | CRISPR-Cas9 long ssDNA donor knock-in endogenous tagging SoxB1 composite tag |
| url | https://www.frontiersin.org/articles/10.3389/fcell.2020.598634/full |
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