在杂交酵母Zygosaccharomyces parabailii中进行CRISPR-Cas9工程可导致靶染色体的杂合性丧失。

IF 2.4 4区 生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Pooja Jayaprakash, Liliane Barroso, Matteo Vajente, Letizia Maestroni, Edward J Louis, John P Morrissey, Paola Branduardi
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引用次数: 0

摘要

杂交酵母Zygosaccharomyces parabailii具有作为细胞工厂的潜力,主要是因为它在承受通常表征生物基过程的应激源方面具有稳健性。然而,复杂的基因组和缺乏基因编辑工具阻碍了改造这种酵母的能力。在这项工作中,我们为Z. parabailii开发了一种CRISPR-Cas9基因编辑系统,该系统允许同时破坏或删除基因的两个等位基因。我们评估了四种不同的gRNA表达系统,包括tRNA、tRNA和核酶或核酶作为自切割侧翼元件的组合,并确定了最有效的系统使用RNA Pol II启动子,然后是gRNA侧翼的5'tRNA。然后利用该gRNA系统构建了一株双链断裂的Z. parabailii菌株,其中DNL4的两个等位基因都失活,因此依靠同源重组来修复双链断裂。我们的系统可以用于野生型菌株的基因失活和dn14突变体的精确缺失与标记插入。在某些情况下,我们观察到DSB位点周围的染色体间重组可能通过基因转换或缺失导致杂合性丧失。虽然这是菌株工程中需要监测的另一个方面,但这种现象也为探索杂交酵母的基因组可塑性提供了机会。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

CRISPR-Cas9 engineering in the hybrid yeast Zygosaccharomyces parabailii can lead to loss of heterozygosity in target chromosomes.

The hybrid yeast Zygosaccharomyces parabailii holds potential as a cell factory mainly because of its robustness in withstanding stressors that often characterize bio-based processes. However, a complex genome and a lack of gene editing tools hinder the capacity to engineer this yeast. In this work, we developed a CRISPR-Cas9 gene editing system for Z. parabailii that allows simultaneous disruption or deletion of both alleles of a gene. We evaluated four different gRNA expression systems consisting of combinations of tRNAs, tRNA and ribozyme or ribozymes as self-cleaving flanking elements and established that the most efficient systems used an RNA Pol II promoter followed by a 5'tRNA flanking the gRNA. This gRNA system was then used to construct a strain of Z. parabailii in which both alleles of DNL4 were inactivated and so relied on homologous recombination to repair double-stranded breaks. Our system can be used for gene inactivation in a wild-type strain and precise deletion with marker insertion in a dnl4 mutant. In some cases, we observed inter-chromosomal recombination around the site of the DSB that could cause loss of heterozygosity through gene conversion or deletion. Although an additional aspect that needs to be monitored during strain engineering, this phenomenon also offers opportunities to explore genome plasticity in hybrid yeasts.

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来源期刊
FEMS yeast research
FEMS yeast research 生物-生物工程与应用微生物
CiteScore
5.70
自引率
6.20%
发文量
54
审稿时长
1 months
期刊介绍: FEMS Yeast Research offers efficient publication of high-quality original Research Articles, Mini-reviews, Letters to the Editor, Perspectives and Commentaries that express current opinions. The journal will select for publication only those manuscripts deemed to be of major relevance to the field and generally will not consider articles that are largely descriptive without insights on underlying mechanism or biology. Submissions on any yeast species are welcome provided they report results within the scope outlined below and are of significance to the yeast field.
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