Genome editing using a DNA‐free clustered regularly interspaced short palindromic repeats‐Cas9 system in green seaweed Ulva prolifera

IF 1.6 4区生物学 Q2 MARINE & FRESHWATER BIOLOGY

Phycological Research Pub Date : 2021-11-03 DOI:10.1111/pre.12472

K. Ichihara, T. Yamazaki, S. Kawano

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引用次数: 6

Abstract

Although the green seaweed Ulva is one of the most common seaweeds in the coastal regions with well‐studied ecological characteristics, few reverse genetic technologies have been developed for it. The clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 system is a simple genome‐editing technology based on a ribonucleoprotein (RNP) complex composed of an endonuclease and programmable RNA to target particular DNA sequences. Genome editing makes it possible to generate mutations on a target gene in non‐model organisms without established transgenic technologies. In this study, we applied the CRISPR‐Cas9 RNP genome‐editing system to the green seaweed Ulva prolifera, using polyethylene glycol (PEG)‐mediated transfection. Our experimental system disrupts a single gene (UpAPT) encoding adenine phosphoribosyl transferase (APT) and generates a resistant phenotype for gametophytes cultured in a medium with toxic compound 2‐fluoroadenine. The PEG‐mediated transfection used for gametes resulted in 2‐fluoroadenine‐resistant strains containing short indels or substitutions on UpAPT. Our results showed that the CRISPR‐Cas9 system with PEG‐mediated transfection was efficient for genome editing in Ulva.

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使用无DNA集群规则间隔短回文重复序列- Cas9系统在绿海藻Ulva prolifera进行基因组编辑

尽管绿色海藻Ulva是沿海地区最常见的海藻之一，具有经过充分研究的生态特征，但几乎没有开发出针对它的反向遗传技术。集群规则间隔短回文重复序列（CRISPR）-Cas9系统是一种简单的基因组编辑技术，基于核糖核蛋白（RNP）复合物，该复合物由核酸内切酶和可编程RNA组成，以靶向特定的DNA序列。基因组编辑使得在没有建立转基因技术的情况下，在非模式生物中产生目标基因突变成为可能。在本研究中，我们使用聚乙二醇（PEG）介导的转染将CRISPR‐Cas9 RNP基因组编辑系统应用于绿色海藻Ulva prolifera。我们的实验系统破坏了编码腺嘌呤磷酸核糖转移酶（APT）的单个基因（UpAPT），并对在含有有毒化合物2-氟腺嘌呤的培养基中培养的配子体产生抗性表型。用于配子的PEG介导的转染导致了在UpAPT上含有短茚或取代的2-氟腺嘌呤抗性菌株。我们的结果表明，具有PEG介导的转染的CRISPR‐Cas9系统对Ulva的基因组编辑是有效的。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Phycological Research 生物-海洋与淡水生物学

CiteScore

3.60

自引率

13.30%

发文量

审稿时长

>12 weeks

期刊介绍： Phycological Research is published by the Japanese Society of Phycology and complements the Japanese Journal of Phycology. The Journal publishes international, basic or applied, peer-reviewed research dealing with all aspects of phycology including ecology, taxonomy and phylogeny, evolution, genetics, molecular biology, biochemistry, cell biology, morphology, physiology, new techniques to facilitate the international exchange of results. All articles are peer-reviewed by at least two researchers expert in the filed of the submitted paper. Phycological Research has been credited by the International Association for Plant Taxonomy for the purpose of registration of new non-vascular plant names (including fossils).