Plant Com | 马里兰大学帕克分校学者开发高效的植物基因组多重碱基编辑工具

以下文章来源于莱肯生物 ，作者莱肯

CRISPR-Cas12a是一种高效的多重基因编辑工具，其对富含T的PAM基序的偏好能与SpCas9形成互补。然而，由于缺乏合适的Cas12a切口酶，基于失活的Cas12a（dCas12a）的植物碱基编辑体系尚未开发出来。

在这项研究中，马里兰大学帕克分校学者开发了植物基因组多重碱基编辑器Cas12a-CBE和Cas12a-ABE。与基于Cas9内切酶的碱基编辑器相比，dCas12a靶向富含T的PAM，这种碱基编辑器产生DNA断裂的可能性较小，非常适合于多重启动子编辑，以微调基因表达而不产生indel突变。基于CRISPR-Cas12a表达系统，研究人员开发了高效的LbCas12a-D156R变体、胞嘧啶脱氨酶和腺嘌呤脱氨酶及其最佳连接体的组合。优化的Cas12a-CBE在高活性靶位点产生高效的单等位基因编辑。值得注意的是，本研究中开发的dLbCas12a-D156R ABE与Cas9 ABE一样，是一种有效的水稻A-to-G碱基编辑系统，在水稻基因组的多个测试靶位点完成编辑，并产生了多个单等位基因和双等位基因编辑系。T0代水稻编辑苗中未检测到明显的脱靶效应。

这些有效的无双链DNA断裂的Cas12a-CBE和Cas12a-ABE编辑器已在Addgene网站上线，将成为植物基因组精准多重碱基编辑的新工具。

PLANT COMMUNICATIONS, 11 April 2023

CRISPR-Cas12a base editors confer efficient multiplexed genome editing in rice

Author

YanhaoCheng, Yingxiao Zhang, Gen Li, Hong Fang, Simon Sretenovic, Avery Fan, Jiang Li, Jianping Xu, Qiudeng Que, Yiping Qi*

*: Department of Plant Science and Landscape Architecture; Institute for Bioscience and Biotechnology Research, University of Maryland, College Park, USA

Abstract

In this study, we developed highly efficient Cas12a CBEs and ABEs for multiplexed genome editing in plants. With less potential to generate DNA breaks compared to Cas9 nickase-based BEs, these T-rich PAM-targeting dCas12a base editors are very suitable for multiplexed promoter editing to fine-tune gene expression without generating indel mutations. Our success is based on the combination of an efficient CRISPR-Cas12a expression system, a high-efficiency LbCas12a-d156R variant, high-activity cytidine and adenine deaminases, and optimal linkers. The optimized Cas12a CBEs produced highly efficient monoallelic editing at high-activity target sites. Significantly, the dLbCas12a-D156R ABEs developed in this study appear to be as efficient as the Cas9 ABEs. These efficient DNA break-free Cas12a CBE and ABE constructs have been deposited to Addgene, which are promising tools for singular and multiplexed base editing in plants.

转自：“植物生物技术Pbj”微信公众号

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