Targeted CRISPR-Cas9-based gene knockouts in the model brown alga Ectocarpus
Targeted CRISPR-Cas9-based gene knockouts in the model brown alga Ectocarpus
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DOI：
10.1111/nph.17525
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发表时间：
2021-07-10
期刊：
影响因子：
9.4
通讯作者：
Cock, J. Mark
中科院分区：
文献类型：
--
作者：
Badis, Yacine;Scornet, Delphine;Cock, J. Mark
褐藻是一类重要的多细胞真核生物，在系统发育上不同于动物和陆地植物谱系。 Ectocarpus 已成为研究褐藻生物学各个方面的模式生物，但该系统目前缺乏有效的反向遗传学方法来分析选定目标基因的功能。在这里，我们报告使用生物射弹或显微注射作为递送方法将 CRISPR-Cas9 核糖核蛋白引入 Ectocarpus 细胞后，在特定靶位点产生突变。在用2-氟腺嘌呤治疗后，分离出具有影响腺嘌呤磷酸核糖基转移酶（APT）基因突变的个体，并且该选择系统用于分离在APT和第二个基因上同时引入突变的个体。这种双突变方法有可能用于分离影响任何 Ectocarpus 基因的突变体，为这种模式生物提供有效的反向遗传学工具。该工具的可用性将显着增强 Ectocarpus 作为这一具有生态和经济重要性的海洋生物群的模式生物的效用。此外，这里描述的方法应该很容易转移到其他褐藻物种。
Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology, but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes. Here, we report that mutations at specific target sites are generated following the introduction of CRISPR-Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method. Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE (APT) gene were isolated following treatment with 2-fluoroadenine, and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism. The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.