在擬南芥中利用CRISPR / Cas9進行超高效基因編輯

在擬南芥中利用CRISPR / Cas9進行超高效基因編輯

 

Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9

https://www.nature.com/articles/s41598-018-22667-1

Julius Durr, Ranjith Papareddy, Keiji Nakajima & Jose Gutierrez-Marcos

doi:10.1038/s41598-018-22667-1

植物生物技術；植物遺傳學

Published:

13 March 2018

Genome editing using CRISPR/Cas9 is considered the best instrument for genome engineering in plants. This methodology is based on the nuclease activity of Cas9 that is guided to specific genome sequences by single guide RNAs (sgRNAs) thus enabling researchers to engineer simple mutations or large chromosomal deletions. Current methodologies for targeted genome editing in plants using CRISPR/Cas9 are however largely inefficient, mostly due to low Cas9 activity, variable sgRNA efficiency and low heritability of genetic lesions. Here, we describe a newly developed strategy to enhance CRISPR/Cas9 efficiency in Arabidopsis thaliana focusing on the design of novel binary vectors (pUbiCAS9-Red and pEciCAS9-Red), the selection of highly efficient sgRNAs, and the use of direct plant regeneration from induced cell cultures. Our work demonstrates that by combining these three independent developments, heritable targeted chromosomal deletions of large gene clusters and intergenic regulatory sequences can be engineered at a high efficiency. Our results demonstrate that this improved CRISPR/Cas9 methodology can provide a fast, efficient and cost-effective tool to engineer targeted heritable chromosomal deletions, which will be instrumental for future high-throughput functional genomics studies in plants.

近期，在《科學報告》上發表的一篇文章Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9中，來自英國華威大學的Jose Gutierrez-Marcos及同事發現了一種利用CRISPR / Cas9進行高效基因編輯的方法。

使用CRISPR / Cas9進行基因組編輯被認為是植物基因組工程的最佳工具。這一方法是基於Cas9的核酸酶活性，單導核酸（sgRNAs）可引導Cas9指向特殊的基因組序列，使研究者能夠對簡單的突變和大型染色體的剪切進行編輯。

然而，目前在植物中使用CRISPR / Cas9進行定點基因組編輯的方法大多效率較低，很大程度上是源於Cas9的活性較低，sgRNA 的效率不定，以及基因修改的遺傳率較低。

在本文中，作者描述了一個新方法，以提高擬南芥中CRISPR/Cas9的編輯效率，該方法著眼於設計新型雙元載體（pUbiCAS9-Red 及pEciCAS9-Red），選取高效sgRNA，及利用細胞培養進行直接植株再生。

作者的研究表明，通過結合上述三方面取得的進展，可高效編輯大型基因簇的可遺傳定點染色體刪減，及基因間可調控序列。作者得到的結果表明這種改進的CRISPR/Cas9法可以快速、高效、經濟地編輯定點可遺傳染色體的刪減，也有助於未來進行高效率功能基因組的研究。（來源：科學網）