利用内源pGhαGloA启动子介导的CRISPR/Cas 12a系统进行高效基因组编辑并创造无腺体棉花种质

doi:10.1016/j.jia.2024.09.011

利用内源pGhαGloA启动子介导的CRISPR/Cas 12a系统进行高效基因组编辑并创造无腺体棉花种质

收稿日期:2024-04-24 修回日期:2024-09-21

Application of an endogenous pGhαGloA promoter in CRISPR/Cas12a system for efficient genome editing to creat glandless cotton germplasm

Chenyu Li^1,^3*, Zumuremu Tuerxun^1*, Yang Yang¹, Xiaorong Li¹, Fengjiao Hui², Juan Li¹ , Zhigang Liu¹, Guo Chen¹, Darun Cai¹, Hui Zhang¹, Xunji Chen¹, Shuangxia Jin^2#, Bo Li^1#

¹Xinjiang Key Laboratory of Crop Biotechnology, The State Key Laboratory of Genetic Improvement and Germplasm Innovation of Crop Resistance in Arid Desert Regions (Preparation), Institute of Nuclear and Biological Technology, Xinjiang Academy of Agricultural Sciences, Urumqi 83000, China
²Hubei Hongshan Laboratory, National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China
³College of Agronomy, Xinjiang Agricultural University, Urumqi 830052, China

Received:2024-04-24 Revised:2024-09-21
About author:#Correspondence Shuangxia Jin, E-mail: jsx@mail.hzau.edu.cn; Bo Li, E-mail: lbharrywei@sina.com * These authors contributed equally to this work.
Supported by:
This study was financially supported by Major Science and Technology Project of Xinjiang Uygur Autonomous Region, China (2023A02003-2) to Bo Li, STI 2030-Major Projects (2023ZD04074) to Dr. Shuangxia Jin, Tianshan Talent training program of Xinjiang Uygur Autonomous Region, China (2023TSYCJU0001), the earmarked fund for XinJiang Agriculture Research System, China (XJARS-3) and the Project of Fund for Stable Support to Agricultural Sci-Tech Renovation (xjnkywdzc-2022001-1) to Bo Li.

摘要/Abstract

摘要：

CRISPR/Cas 12a系统是一种高效的基因组编辑工具，在植物功能基因组学研究和农艺性状改良中得到了广泛应用。本研究利用棉花内源pGhαGloA启动子对CRISPR/Cas 12a系统进行了优化。利用该系统，在Pol II类型的pGhaGloA启动子的驱动下，构建了pGhRBE3-pGhαGloA-GhPGF载体，并进行了遗传转化。该载体在所有阳性转基因植株中均能有效工作，crRNA1靶位点的编辑效率高达93.37%，crRNA2靶位点的编辑效率高达88.24%，明显高于Pol III启动子-Ubi 6.7驱动的的pGhRBE3系统的编辑效率，表明Pol II启动子比Pol III启动子更适合于在棉花中表达多种sgRNA或crRNA。该载体的主要编辑类型呈现片段缺失，缺失片段大小在3-12 bp之间，编辑位点位于PAM下游第14 ~ 29位碱基。这些突变位点在T₀到T₂代均能稳定遗传，同时获得了3个GhPGF基因突变的DNA-Free株系，这些无棉酚或低酚棉花种质将对棉籽油/饼的健康生产起重要用。因此，pGhαGloA启动子驱动的CRISPR/Cas 12a系统可以在棉花中高效编辑靶标基因，为棉花功能基因组学和遗传改良提供了有力的工具。

Abstract:

The efficient genome editing tool (the CRISPR/Cas12a system) has been used in research on plant functionional genomics and improvement of agronomic traits. In this study, CRISPR/Cas12a system was optimized by using the endogenous pGhαGloA promoter in cotton. Using this system, crRNAs was driven by the Pol II pGhaGloA promoter to construct the pGhRBE3-pGhαGloA-GhPGF vector and carry out genetic transformation. The vector could work efficiently in all positive transgenic plants and the editing efficiency at the crRNA1 target site was up to 93.37%, and the editing efficiency of the crRNA2 target was up to 88.24%, which is significantly higher in editing efficiency of the pGhRBE3 system with Pol III promoter-Ubi 6.7 promoter, this result indicates that the Pol II promoter is more suitable for expressing multiple sgRNA or crRNA than the pol III promoter in cotton. The vector mainly generated the editing type of fragment deletion and the deletion size was in the range of 3-12 bp with the editing sites spanning at the 14th to 29th bases downstream of the protospacer adjacent motif (PAM). All the targeted mutation loci were stably inherited from T₀ to T₂ generation and three transgene-free lines with target site mutations of GhPGF gene were obtained and these glandless and gossypol-free/(low contents) cotton germplasm will play key role for healthy cottonseeds oil/cake production. Therefore, the CRISPR/Cas12a system driven by the pGhαGloA promoter can efficiently edit target genes in cotton, which provides a powerful tool for cotton functionional genomics and genetic improvement.

Key words: cotton , genome editing , , CRISPR/Cas12a , , Pol II promoter , , glandless cotton , , gossypol-free

. 利用内源pGhαGloA启动子介导的CRISPR/Cas 12a系统进行高效基因组编辑并创造无腺体棉花种质[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.09.011.

Chenyu Li, Zumuremu Tuerxun, Yang Yang, Xiaorong Li, Fengjiao Hui, Juan Li, Zhigang Liu, Guo Chen, Darun Cai, Hui Zhang, Xunji Chen, Shuangxia Jin, Bo Li. Application of an endogenous pGhαGloA promoter in CRISPR/Cas12a system for efficient genome editing to creat glandless cotton germplasm[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2024.09.011.

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