Application of CRISPR-Cas9 genome editing by microinjection of gametophytes of Saccharina japonica (Laminariales, Phaeophyceae)
Application of CRISPR-Cas9 genome editing by microinjection of gametophytes of Saccharina japonica (Laminariales, Phaeophyceae)
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通过显微注射海带目、褐藻纲配子体进行 CRISPR-Cas9 基因组编辑的应用
DOI：
10.1007/s10811-023-02940-1
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发表时间：
2023
影响因子：
3.3
通讯作者：
Nagasato Chikako
中科院分区：
文献类型：
--
作者：
Shen Yuan;Motomura Taizo;Ichihara Kensuke;Matsuda Yusuke;Yoshimura Ko;Kosugi Chika;Nagasato Chikako
规则间隔短回文重复序列（CRISPR）-Cas9介导的基因组编辑已用于许多生物体的反向遗传学研究。然而，应用于商业上重要的海藻物种仍然有限。通过开发基因组编辑工具，将推进海带目物种的遗传和育种技术。在这项研究中，我们尝试使用CRISPR-Cas9核糖核蛋白（RNP）复合物通过显微注射到日本野牡丹配子体中来编辑反向选择标记，腺嘌呤磷酸核糖转移酶（APT）基因。注射CRISPR-Cas9 RNP后，向培养基中加入2-氟腺嘌呤（2-FA）（10-40 μM）以选择APT突变体。23个雌性和12个雄性2-FA抗性配子体的APT基因发生突变。注射试验的基因组编辑效率对于雌性和雄性配子体分别为8.64%和4.46%。突变配子体能够产生精子或卵子。突变配子体的精卵杂交产生的孢子体对40 μM 2-FA有抗性。来自一个野生型和一个突变亲本杂交的孢子体也显示出对高浓度（20-40 μM）2-FA的抗性，而野生型孢子体在10 μM 2-FA中死亡。这是第一份显示S基因编辑有效性的报告。使用CRISPR-Cas9 RNP复合物和显微注射方法，
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated genome editing has been used for reverse genetics studies in many organisms. However, application to commercially important species of seaweeds is still limited. The genetics and breeding technologies for species of the Laminariales will be advanced by developing a genome editing tool. In this study, we attempted to edit a counter-selectable marker, theadenine phosphoribosyl transferase(APT) gene using CRISPR-Cas9 ribonucleoprotein (RNP) complexes delivered by microinjection into gametophytes ofSaccharina japonica. After injection of CRISPR-Cas9 RNP, 2-fluoroadenine (2-FA) was added to the medium (10–40 μM) to selectAPTmutants. Twenty-three female and 12 male 2-FA resistant gametophytes had mutations in theirAPTgenes. Genome editing efficiency for the injection trials was 8.64% and 4.46% for the female and male gametophytes, respectively. Theaptmutant gametophytes were able to produce sperm or eggs. Sporophytes derived by crossing sperm and eggs fromaptmutant gametophytes were resistant to 40 μM 2-FA. Sporophytes derived from crosses with one wild type and one mutant parent also showed resistance to high concentrations (20–40 μM) of 2-FA, while wild-type sporophytes died in 10 μM 2-FA. This is the first report showing the validity of gene editing ofS. japonicausing the CRISPR-Cas9 RNP complex and microinjection method.
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DOI：
10.1073/pnas.1610725113
发表时间：
2016-11
期刊：
Proceedings of the National Academy of Sciences
影响因子：
--
作者：
A. Vergés;C. Doropoulos;H. Malcolm;Mathew Skye;Marina Garcia-Pizá;E. Marzinelli;A. Campbell;E. Ballesteros;A. Hoey;A. Vila‐Concejo;Y. Bozec;P. Steinberg
通讯作者：
A. Vergés;C. Doropoulos;H. Malcolm;Mathew Skye;Marina Garcia-Pizá;E. Marzinelli;A. Campbell;E. Ballesteros;A. Hoey;A. Vila‐Concejo;Y. Bozec;P. Steinberg
培养基中铁浓度调节海带（Laminariales，Phaeophyta）配子发生的超微结构研究
影响因子：
1.6
作者：
T. Motomura;Y. Sakai
通讯作者：
Y. Sakai
影响因子：
9.4
作者：
Badis, Yacine;Scornet, Delphine;Cock, J. Mark
通讯作者：
Cock, J. Mark
通过实时 PCR 定量海水中的海带游动孢子
影响因子：
1.5
作者：
Chikako Nagasato;H. Kawamoto;T. Tomioka;S. Tsuyuzaki;Chika Kosugi;Toshiaki Kato;T. Motomura
通讯作者：
T. Motomura
影响因子：
2.5
作者：
Christie, Hartvig;Norderhaug, Kjell Magnus;Fredriksen, Stein
通讯作者：
Fredriksen, Stein