Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration.
Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration.
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DOI：
10.1111/pbi.12870
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发表时间：
2018-07
影响因子：
13.8
通讯作者：
Shih MC
中科院分区：
文献类型：
--
作者：
Lin CS;Hsu CT;Yang LH;Lee LY;Fu JY;Cheng QW;Wu FH;Hsiao HC;Zhang Y;Zhang R;Chang WJ;Yu CT;Wang W;Liao LJ;Gelvin SB;Shih MC
植物原生质体可用于评估成簇规则间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）诱变的效率。我们改进了几种植物的原生质体分离和转染的方法。我们还开发了一种分离和再生单一诱变烟草原生质体到成熟植株的方法。在用编码Cas9和sgRNA的构建体转染原生质体后，可以扩增靶基因DNA用于进一步分析以确定诱变效率。我们研究了烟草原生质体和再生植株的八氢番茄红素去饱和酶（NtPDS）基因的定向诱变。白化病再生植株的基因分型表明，在双二倍体烟草中所有四个NtPDS等位基因都发生了突变，并且在大多数再生植株中不能检测到Cas9 DNA。
Plant protoplasts are useful for assessing the efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) mutagenesis. We improved the process of protoplast isolation and transfection of several plant species. We also developed a method to isolate and regenerate single mutagenized Nicotianna tabacum protoplasts into mature plants. Following transfection of protoplasts with constructs encoding Cas9 and sgRNAs, target gene DNA could be amplified for further analysis to determine mutagenesis efficiency. We investigated N. tabacum protoplasts and derived regenerated plants for targeted mutagenesis of the phytoene desaturase (NtPDS) gene. Genotyping of albino regenerants indicated that all four NtPDS alleles were mutated in amphidiploid tobacco, and no Cas9 DNA could be detected in most regenerated plants.
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