一种适用于CRISPR/Cas基因编辑技术的gRNA预筛选瞬时转化策略

doi:10.1016/j.jia.2025.04.007

一种适用于CRISPR/Cas基因编辑技术的gRNA预筛选瞬时转化策略

修回日期:2025-04-03

A transient transformation method for pre-screening of gRNAs in CRISPR/Cas gene editing

Jinghui Xu¹^*, Xiaocui Yan⁴^*, Yan Yu¹^*, Hang Zhao^2,³^*, Menghan Liu², Ye Wang², Peng Wang², Hongying Duan¹^#, Xiaoyang Ge²^#, Jingjing Zhan²^#

¹Henan Normal University Research Base of National Key Laboratory of Cotton Bio-breeding and Integrated Utilization, Xinxiang 453000, China
²National Key Laboratory of Cotton Bio-breeding and Integrated Utilization/Institute of Cotton Research, Chinese Academy of Agricultural Sciences, Anyang 455000, China
³College of Life Sciences, Qufu Normal University, Qufu 273100, China
⁴State Key Laboratory of North China Crop Improvement and Regulation Key Germplasm Resources in North China, Ministry of Education, College of Agronomy, Hebei Agricultural University, Baoding 071001, China

Revised:2025-04-03
About author:#Correspondence Xiaoyang Ge, E-mail: gexiaoyang@caas.cn; Jingjing Zhan, E-mail: jjzhan0925@126.com; Hongying Duan, E-mail: dxdhy@126.com
Supported by:
This work was supported by the Agricultural GongGuan Project of Xinjiang Production and Construction Corps, China (NYHXGG, 2023AA102), the Innovation Program of Chinese Academy of Agricultural Sciences (CAAS-CSIAF-202402), the Outstanding Youth Foundation of Henan Scientific Committee, China (222300420097), and the China Agriculture Research System of MOF and MARA (CARS-15-02).

摘要/Abstract

摘要：

基因编辑系统在阐明植物基因功能和促进分子设计育种方面具有广泛的潜力。然而，单引导rna（sgRNAs）的效率各不相同，通过生物信息学准确预测其效率仍然存在挑战，特别是在棉花（陆地棉）等作物中。在本研究中，我们开发了一种快速、有效的方法，利用一个瞬时表达系统来验证棉花中sgRNAs的功能，它可以在三天内完成。本研究选择6个基因12个靶点，观察到通过稳定和瞬态转换获得的编辑效率呈正相关，皮尔逊相关系数（R2）为0.71。我们的研究通过评估多个基因的不同gRNA序列的效率，证实该方法可以快速评估gRNA对基因组的编辑效率，从而通过预筛选提高基因编辑的工作效率。

. 一种适用于CRISPR/Cas基因编辑技术的gRNA预筛选瞬时转化策略[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2025.04.007.

Jinghui Xu, Xiaocui Yan, Yan Yu, Hang Zhao, Menghan Liu, Ye Wang, Peng Wang, Hongying Duan, Xiaoyang Ge, Jingjing Zhan. A transient transformation method for pre-screening of gRNAs in CRISPR/Cas gene editing[J]. Journal of Integrative Agriculture, DOI: 10.1016/j.jia.2025.04.007.

参考文献

Bortesi L, Fischer R. 2015. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnology Advances, 33, 41-52.

Cardi T, Murovec J, Bakhsh A, Boniecka J, Bruegmann T, Bull S E, Eeckhaut T, Fladung M, Galovic V, Linkiewicz A, Lukan T, Mafra I, Michalski K, Kavas M, Nicolia A, Nowakowska J, Sági L, Sarmiento C, Yıldırım K, Zlatković M, Hensel G, Laere K V. 2023. CRISPR/Cas-mediated plant genome editing: Outstanding challenges a decade after implementation. Trends in Plant Science, 28, 1144-1165.

Cong L, Ran F A, Cox D, Lin S, Barretto R, Habib N, Hsu P D, Wu X, Jiang W, Marraffini L A, Zhang F. 2013. Multiplex genome engineering using CRISPR/Cas systems. Science, 339, 819-823.

Gao W, Long L, Tian X, Xu F, Liu J, Singh P K, Botella J R, Song C. 2017. Genome editing in cotton with the CRISPR/Cas9 system. Frontiers in Plant Science, 8, 1364.

Gao X, Wheeler T, Li Z, Kenerley C M, He P, Shan L. 2011. Silencing GhNDR1 and GhMKK2 compromises cotton resistance to Verticillium wilt. Plant Journal, 66, 293-305.

Ge X, Xu J, Yang Z, Yang X, Wang Y, Chen Y, Wang P, Li F. 2023. Efficient genotype-independent cotton genetic transformation and genome editing. Journal of Integrative Plant Biology, 65, 907-917.

Lin Q, Zong Y, Xue C, Wang S, Jin S, Zhu Z, Wang Y. 2020. Prime genome editing in rice and wheat. Nature Biotechnology, 38, 582-585.

Liu Q, Wang C, Jiao X, Zhang H, Song L, Li Y, Gao C, Wang K. 2019. Hi-TOM: A platform for high-throughput tracking of mutations induced by CRISPR/Cas systems. Science China (Life Sciences), 62, 1-7.

Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu Y. 2015. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Molecular Plant, 8, 1274-1284.

Mussolino C, Cathomen T. 2013. RNA guides genome engineering. Nature Biotechnology, 31, 208-209.

Ran F A, Hsu P D, Wright J, Agarwala V, Scott D A, Zhang F. 2013. Genome engineering using the CRISPR-Cas9 system. Nature Protocols, 8, 2281-2308.

Shan Q, Wang Y, Li J, Gao C. 2014. Genome editing in rice and wheat using the CRISPR/Cas system. Nature Protocols, 9, 2395-2410.

Yang Y, Xu C, Shen Z, Yan C. 2021. Crop quality improvement through genome editing strategy. Frontiers in Genome Editing, 3, 819687.

Zhang K, Raboanatahiry N, Zhu B, Li M. 2017. Progress in genome editing technology and its application in plants. Frontiers in Plant Science, 8, 177.

Zhang Y, Ma X, Xie X, Liu Y G. 2017. CRISPR/Cas9-based genome editing in plants. Progress in Molecular Biology and Translational Science, 149, 133-150.