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

植物生物技术；植物遗传学

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《科学报告》上发表的一篇文章Highly efficient heritable targeted deletions of gene clusters and non-coding regulatory regions in Arabidopsis using CRISPR/Cas9中，来自英国华威大学的Jose Gutierrez-Marcos及同事发现了一种利用CRISPR / Cas9进行高效基因编辑的方法。

使用CRISPR / Cas9进行基因组编辑被认为是植物基因组工程的最佳工具。这一方法是基于Cas9的核酸酶活性，单导核酸（sgRNAs）可引导Cas9指向特殊的基因组序列，使研究者能够对简单的突变和大型染色体的剪切进行编辑。

然而，目前在植物中使用CRISPR / Cas9进行定点基因组编辑的方法大多效率较低，很大程度上是源于Cas9的活性较低，sgRNA 的效率不定，以及基因修改的遗传率较低。

在本文中，作者描述了一个新方法，以提高拟南芥中CRISPR/Cas9的编辑效率，该方法着眼于设计新型双元载体（pUbiCAS9-Red 及pEciCAS9-Red），选取高效sgRNA，及利用细胞培养进行直接植株再生。

作者的研究表明，通过结合上述三方面取得的进展，可高效编辑大型基因簇的可遗传定点染色体删减，及基因间可调控序列。作者得到的结果表明这种改进的CRISPR/Cas9法可以快速、高效、经济地编辑定点可遗传染色体的删减，也有助于未来进行高效率功能基因组的研究。（来源：科学网）