CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans
CRISPR/Cas9-mediated efficient genome editing via protoplast-based transformation in yeast-like fungus Aureobasidium pullulans
复制标题
通过类酵母真菌出芽短梗霉的原生质体转化，CRISPR/Cas9介导的高效基因组编辑
DOI：
10.1016/j.gene.2019.04.079
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发表时间：
2019
期刊：
影响因子：
3.5
通讯作者：
Zou Xiang
中科院分区：
文献类型：
--
作者：
Zhang Yuan;Feng Jun;Wang Pan;Xia Jun;Li Xiaorong;Zou Xiang
出芽短梗霉是一种环境适应性很强的酵母样真菌，是生物生产各种有价值代谢物的潜在宿主。然而，低效的基因操作限制了其潜在的应用。本研究建立了CRISPR/Cas9介导的原生质体转化基因组编辑系统。为了检测CRISPR/Cas9介导的基因组突变，将5-磷酸鸟苷脱羧酶(UMPS)基因作为反选择选择标记。CRISPR/Cas9系统通过共转化两个分别携带Cas9基因的质粒和一个靶向肿瘤的引导RNA，可显著提高引导RNA靶点的突变频率。为了进一步验证CRISPR/Cas9刺激与供体DNA的同源重组，建立了β-葡萄糖苷酸酶(GUS)基因的颜色报告系统，用于计算阳性突变率。结果表明，CRISPR/Cas9系统的阳性突变率为~40%，明显高于单纯供体DNA的阳性突变率(~4%)。此外，通过比较侧翼gRNA和自切核酶或tRNA的效果，分析了不同的转录后RNA处理方案。结果表明，用自切割核酶处理的gRNA具有较高的阳性突变率。本研究为一种简单、功能强大的出芽短梗霉基因组编辑工具奠定了基础。此外，还开发了反选择选择标记(Umps)和颜色报告系统(Gus)，作为菌株工程的遗传部分。
Aureobasidium pullulans, a yeast-like fungus with strong environmental adaptability, remains a potential host for bio-production of different valuable metabolites. However, its potential application is limited by low-efficient genetic manipulation. In this study, CRISPR/Cas9-mediated genome editing via protoplast-based transformation system was developed. To test CRISPR/Cas9 mediated genomic mutagenesis, the orotidine 5-phosphate decarboxylase (umps) gene was used as a counter-selectable selection marker. By co-transforming of two plasmids harboringcas9gene and a guide RNA targetingumps, respectively, the CRISPR/Cas9 system could significantly increase frequency of mutation in the targeting site of guide RNA. To further validate that CRISPR/Cas9 stimulated homologous recombination with donor DNA, a color reporter system of beta-glucuronidase (gus) gene was developed for calculating positive mutation rate. The results showed that positive mutation rate with CRISPR/Cas9 system was ~40% significantly higher than only with the donor DNA (~4%). Furthermore, the different posttranscriptional RNA processing schemes were analyzed by compared the effects of flanking gRNA with self-cleaving ribozymes or tRNA. The result demonstrated that gRNA processed by self-cleaving ribozymes achieves higher positive mutant rate. This study provided foundation for a simple and powerful genome editing tool forA.pullulans. Moreover, a counter-selectable selection marker (umps) and a color reporter system (gus) were being developed as genetic parts for strain engineering.