Trans-nuclei CRISPR/Cas9: safe approach for genome editing in the edible mushroom excluding foreign DNA sequences

IF 3.9 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Daishiro Koshi, Junko Sugano, Fuga Yamasaki, Moriyuki Kawauchi, Takehito Nakazawa, Minji Oh, Yoichi Honda
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引用次数: 0

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions. In this study, we developed a foreign-DNA-free genome editing method in Pleurotus ostreatus by transferring the Cas9/guide RNA (gRNA) complex between nuclei in the dikaryotic state. We isolated a donor monokaryotic P. ostreatus strain expressing Cas9 and gRNA targeting pyrG by introducing a recombinant plasmid, which exhibited uracil auxotrophy and 5-fluoroorotic acid (5-FOA) resistance. This strain was then crossed with a pyrG+ recipient monokaryon, resulting in dikaryotic strains exhibiting 5-FOA resistance after mycelial growth. When these strains were de-dikaryonized into monokaryons through protoplasting, we obtained monokaryotic isolates harboring the recipient nucleus with small indels at the pyrG target site. Importantly, these isolates were confirmed to be free of foreign DNA through genomic PCR, Southern blotting, and whole-genome resequencing analyses. This is the first report of an efficient genome editing protocol in agaricomycetes that ensures no integration of exogenous DNA. This approach is expected to be applicable to other fungi with a dikaryotic life cycle, opening new possibilities for molecular breeding without the concerns associated with GMOs.

• Successful genome editing via CRISPR/Cas9 trans-nuclei manner in P. ostreatus.

Recipient monokaryons from gene-edited dikaryons showed no exogenous DNA sequences.

Efficient genome editing protocol for safer molecular breeding in mushroom fungus.

跨核CRISPR/Cas9:排除外源DNA序列的食用菌基因组编辑安全方法
簇状规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9 (Cas9)辅助基因组编辑已应用于几种主要的食用木丝菌,实现了高效的基因靶向。该方法为快速高效地分离高价值品种和克服栽培挑战提供了有利条件。然而,在这一过程中外源DNA片段的整合引起了人们对转基因生物及其监管限制的关注。在本研究中,我们通过在双核状态下在细胞核间转移Cas9/向导RNA (gRNA)复合物,建立了一种无外源dna的平菇(Pleurotus ostreatus)基因组编辑方法。我们通过引入重组质粒分离到了一株表达Cas9和靶向pyrG的gRNA的供体单核P. ostreatus菌株,该菌株表现出尿嘧啶萎缩和5-氟乙酸(5-FOA)抗性。然后将该菌株与pyg +受体单核细胞杂交,得到在菌丝生长后表现出5-FOA抗性的双核菌株。当这些菌株通过原生质体去二核化为单核时,我们获得了在pyrG靶位点具有小indeks的受体细胞核的单核分离株。重要的是,通过基因组PCR、Southern blotting和全基因组重测序分析,这些分离株被证实不含外源DNA。这是第一个有效的琼脂菌基因组编辑方案,确保不整合外源DNA的报告。这种方法有望适用于其他具有双核生命周期的真菌,为分子育种开辟新的可能性,而无需担心与转基因生物相关的问题。•通过CRISPR/Cas9反核方式成功编辑P. ostreatus基因组。•来自基因编辑二核子的受体单核子未显示外源DNA序列。•高效的基因组编辑方案,更安全的蘑菇分子育种。
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来源期刊
Applied Microbiology and Biotechnology
Applied Microbiology and Biotechnology 工程技术-生物工程与应用微生物
CiteScore
10.00
自引率
4.00%
发文量
535
审稿时长
2 months
期刊介绍: Applied Microbiology and Biotechnology focusses on prokaryotic or eukaryotic cells, relevant enzymes and proteins; applied genetics and molecular biotechnology; genomics and proteomics; applied microbial and cell physiology; environmental biotechnology; process and products and more. The journal welcomes full-length papers and mini-reviews of new and emerging products, processes and technologies.
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