CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus.
CRISPR-Cas9 ribonucleoprotein-mediated co-editing and counterselection in the rice blast fungus.
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DOI：
10.1038/s41598-018-32702-w
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发表时间：
2018-09-25
影响因子：
4.6
通讯作者：
Talbot NJ
中科院分区：
文献类型：
--
作者：
Foster AJ;Martin-Urdiroz M;Yan X;Wright HS;Soanes DM;Talbot NJ
稻瘟病菌是栽培稻上最严重的病原菌，对全球粮食安全构成重大威胁。为了加速该物种的靶向突变和特异性基因组编辑，我们开发了一种快速无质粒的基于CRISPR-Cas9的基因组编辑方法。我们表明Cas9的稳定表达对M.米。然而，有效的基因编辑可以通过瞬时引入预复合到RNA向导的纯化的Cas9以形成核糖核蛋白（RNP）来实现。当与寡核苷酸或PCR生成的供体DNA组合使用时，具有特定碱基对编辑、基因座内基因置换或多个基因编辑的菌株的生成非常快速和直接。我们展示了一种用于在特定位点创建单核苷酸变化的共同编辑策略。此外，我们报告了一种新的反选择策略，该策略允许创建精确编辑的不含外源DNA并且与野生型完全等基因的真菌菌株。总之，这些发展代表了M中遗传操作的精度和速度的可扩展改进。这可能是广泛适用于其他真菌物种。
The rice blast fungus Magnaporthe oryzae is the most serious pathogen of cultivated rice and a significant threat to global food security. To accelerate targeted mutation and specific genome editing in this species, we have developed a rapid plasmid-free CRISPR-Cas9-based genome editing method. We show that stable expression of Cas9 is highly toxic to M. oryzae. However efficient gene editing can be achieved by transient introduction of purified Cas9 pre-complexed to RNA guides to form ribonucleoproteins (RNPs). When used in combination with oligonucleotide or PCR-generated donor DNAs, generation of strains with specific base pair edits, in-locus gene replacements, or multiple gene edits, is very rapid and straightforward. We demonstrate a co-editing strategy for the creation of single nucleotide changes at specific loci. Additionally, we report a novel counterselection strategy which allows creation of precisely edited fungal strains that contain no foreign DNA and are completely isogenic to the wild type. Together, these developments represent a scalable improvement in the precision and speed of genetic manipulation in M. oryzae and are likely to be broadly applicable to other fungal species.
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通讯作者：
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