Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System
Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System
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DOI：
10.1093/pcp/pcw173
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发表时间：
2016-12-01
影响因子：
4.9
通讯作者：
Sakakibara, Hitoshi
中科院分区：
文献类型：
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作者：
Nomura, Toshihisa;Sakurai, Tetsuya;Sakakibara, Hitoshi
由簇状规则间隔短回文重复序列(CRISPR)/CRISPR相关核酸酶9(Cas9)系统衍生的RNA引导的核酸酶靶向基因组修饰在包括几个植物物种在内的许多生物中得到了迅速的发展。在本研究中，我们成功地将CRISPR/Cas9系统引入了非模式生物Scopelophila cataractae(一种具有重金属耐受性的苔藓)和模式生物Physcomitrella patens。利用苔藓植物从原生质体再生的过程，我们通过在原生质体中表达单链引导RNA(SgRNA)和Cas9进行了定向突变。利用这种方法，与目标基因相关的表型变化的菌株的获取率约为45%-69%，表型变化的菌株表现出各种插入和缺失突变。此外，我们报告说，我们的方法能够多重定向突变(两个独立的基因)，并允许有效地引入大的缺失(类似于3KBP)。这些结果表明，CRISPR/CAS9系统可用于加快苔藓学和陆地植物进化的研究。
Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens. Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (similar to 3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution.