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

Chenyu Li1, 3*, Zumuremu Tuerxun1*, Yang Yang1, Xiaorong Li1, Fengjiao Hui2, Juan Li1 , Zhigang Liu1, Guo Chen1, Darun Cai1, Hui Zhang1, Xunji Chen1, Shuangxia Jin2#, Bo Li1#

1 Xinjiang Key Laboratory of Crop BiotechnologyThe State Key Laboratory of Genetic Improvement and Germplasm Innovation of Crop Resistance in Arid Desert Regions (Preparation)Institute of Nuclear and Biological TechnologyXinjiang Academy of Agricultural Sciences, Urumqi 83000, China

2 Hubei Hongshan LaboratoryNational Key Laboratory of Crop Genetic ImprovementHuazhong Agricultural UniversityWuhan 430070, China

3 College of Agronomy, Xinjiang Agricultural University, Urumqi 830052, China

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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sgRNAcrRNA。该载体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T0T2代均能稳定遗传,同时获得了3GhPGF基因突变的DNA-Free系,这些无棉酚或低酚棉花种质将对棉籽油/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.

Keywords:  cotton       genome editing              CRISPR/Cas12a              Pol II promoter              glandless cotton              gossypol-free  
Received: 24 April 2024   Online: 21 September 2024  
Fund: 

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.

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.

Cite this article: 

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. 2024. Application of an endogenous pGhαGloA promoter in CRISPR/Cas12a system for efficient genome editing to creat glandless cotton germplasm. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.09.011

Altae-Tran H, Kannan S, Demircioglu F E, Oshiro R, Nety S P, McKay L J, Dlakić M, Inskeep W P, Makarova K S, Macrae R K, Koonin E V, Zhang F. 2021. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. Science, 374, 57-65.

Bak R O, Gomez-Ospina N, Porteus M H. 2018. Gene editing on center stage. Trends in Genetics, 34, 600-611.

Bello E I, Aladesuru A A. 2015. Cottonseed (Gossypium arboretum) oil biodiesel. Scientia Agriculturae, 11, 1-7.

Cai Y F, Xie Y F, Liu J G. 2010. Glandless seed and glanded plant research in cotton. A review. Agronomy for Sustainable Development, 30, 181-190.

Cao G, Dong J, Chen X, Lu P, Xiong Y F, Peng L, Li J W, Huo D Q, Hou C J. 2022. Simultaneous detection of CaMV35S and T-nos utilizing CRISPR-Cas12a and Cas13a with multiplex-PCR (MPT-Cas12a/13a). Chemical Communications, 58, 6328-6331.

Čermák T, Curtin S J, Gil-Humanes J, Čegan R, Kono T J Y, Konečná E, Belanto J J, Starker C G, Mathre J W, Greenstein R L, Voytas D F. 2017. A multipurpose toolkit to enable advanced genome engineering in plants. The Plant Cell, 29, 1196-1217.

Chuang W Y, Lin L J, Shih H D, Shy Y M, Chang S C, Lee T T. 2021. The potential utilization of high-fiber agricultural by-products as monogastric animal feed and feed additives: A review. Animals, 11, 2098.

Connors B J, Miller M, Maynard C A, Powell W A. 2002. Cloning and characterization of promoters from American chestnut capable of directing reporter gene expression in transgenic Arabidopsis plants. Plant Science, 163, 771-781.

Dharmendra S K, Naimisha C, Kumar A K, Yogendra K, Chikkaputtaiah C, Reddy P S. 2023. RNA Pol III promoters—key players in precisely targeted plant genome editing. Frontiers in Genetics, 13, 989199.

Gao X P, Guo H H, Zhang Q, Guo H X, Zhang L, Zhang C Y, Gou Z Y, Liu Y, Wei J M, Chen A Y, Chu Z H, Zeng F C. 2020. Arbuscular mycorrhizal fungi (AMF) enhanced the growth, yield, fiber quality and phosphorus regulation in upland cotton (Gossypium hirsutum L.). Scientific Reports, 10, 2084.

Ghezraoui H, Piganeau M, Renouf B, Renaud J B, Sallmyr A, Ruis B, Oh S, Tomkinson A E, Hendrickson E A, Giovannangeli C, Jasin M, Brunet E. 2014. Chromosomal translocations in human cells are generated by canonical nonhomologous end-joining. Molecular Cell, 55, 829-842.

Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna J A, Charpentier E. 2012. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816-821.

Joung J K, Sander J D. 2013. TALENs: A widely applicable technology for targeted genome editing. Nature Reviews Molecular Cell Biology, 14, 49-55.

Kamburova V S, Nikitina E V, Shermatov S E, Buriev Z T, Kumpatla S P, Emani C, Abdurakhmonov I Y. 2017. Genome editing in plants: An overview of tools and applications. International Journal of Agronomy, 2017, 1-15.

Kim D, Kim J, Hur J K, Been K W, Yoon S, Kim J S. 2016. Genome-wide analysis reveals specificities of Cas12a endonucleases in human cells. Nature Biotechnology, 34, 863-868.

Kleinstiver B P, Tsai S Q, Prew M S, Nguyen N T, Welch M M, Lopez J M, McCaw Z R, Aryee M J, Joung J K. 2016. Genome-wide specificities of CRISPR-Cas Cas12a nucleases in human cells. Nature Biotechnology, 34, 869-874.

Kumar M, Tomar M, Punia S, Grasso S, Arrutia F, Choudhary J, Singh S, Verma P, Mahapatra A, Patil S, Dhumal S, Potkule J, Saxena S, Amarowicz R. 2021. Cottonseed: A sustainable contributor to global protein requirements. Trends in Food Science & Technology, 111, 100-113.

Li B, Liang S J, Alariqi M, Wang F Q, Wang G Y, Wang Q Q, Xu Z P, Yu L, Zafar M N, Sun L, Si H, Yuan D J, Guo W F, Wang Y Q, Lindsey K, Zhang X L, Jin S X. 2020. The application of temperature sensitivity CRISPR/LbCpf1 (LbCas12a) mediated genome editing in allotetraploid cotton (G. hirsutum) and creation of nontransgenic, gossypol-free cotton. Plant Biotechnology Journal, 19, 221-223.

Li B, Rui H P, Li Y J, Wang Q Q, Alariqi M, Qin L, Sun L, Ding X, Wang F Q, Zou J W, Wang Y Q, Yuan D J, Zhang X L, Jin S X. 2019. Robust CRISPR-Cas12a (Cas12a)‐mediated genome editing in allotetraploid cotton (Gossypium hirsutum). Plant Biotechnology Journal, 17, 1862-1864. 

Li J Y, Manghwar H, Sun L, Wang P C, Wang G Y, Sheng H Y, Zhang J, Liu H, Qin L, Rui H P, Li B, Lindsey K, Daniell H, Jin S X, Zhang X L. 2019. Whole genome sequencing reveals rare off‐target mutations and considerable inherent genetic or/and somaclonal variations in CRISPR/Cas9‐edited cotton plants. Plant Biotechnology Journal, 17, 858-868.

Lin H, Li G, Peng X, Deng A, Ye L, Shi L, Wang T, He J. 2021. The use of CRISPR/Cas9 as a tool to study human infectious viruses. Frontiers in Cellular and Infection Microbiology, 11, 590989.

Ma M R, Yang L, Hu Z Z, Mo C J, Geng S Y, Zhao X, He Q Y, Xiao L, Lu L R, Wang D, Li S G, Kong Q S, Li D W, Bie Z L. 2024. Multiplex gene editing reveals cucumber MILDEW RESISTANCE LOCUS O family roles in powdery mildew resistance. Plant Physiology, 195, 1069-1088..

Mageshwaran V. 2021. An overview of gossypol and methods of its detoxification in cottonseed meal for non-ruminant feed applications. Indian Journal of Natural Products and Resources (IJNPR)[Formerly Natural Product Radiance (NPR)], 12, 348-358.

Mahfouz M M. 2017. Genome editing: The efficient tool CRISPR-Cas12a. Nature Plants, 3, 1-2.

Manghwar H, Lindsey K, Zhang X L, Jin S X. 2019. CRISPR/Cas system: Recent advances and future prospects for genome editing. Trends in Plant Science, 24, 1102-1125.

Mao Y F, Botella J R, Liu Y, Zhu J K. 2019. Gene editing in plants: Progress and challenges. National Science Review, 6, 421-437.

Mikami M, Toki S, Endo M. 2017. In planta processing of the SpCas9–gRNA complex. Plant and Cell Physiology, 58, 1857-1867.

Nagalakshmi D, Rao S V R, Panda A K, Sastry V R B. 2007. Cottonseed meal in poultry diets: A review. The Journal of Poultry Science, 44, 119-134.

Qin L, Li J Y, Wang Q, Xu Z P, Sun L, Alariqi M, Manghwar H, Wang G Y, Li B, Ding X, Rui H P, Huang H M, Lu T L, Lindsey K, Daniell H, Zhang X L, Jin S X. 2020. High-efficient and precise base editing of C•G to T•A in the allotetraploid cotton (Gossypium hirsutum) genome using a modified CRISPR/Cas9 system. Plant Biotechnology Journal, 18, 45-56.

Radhika E, Kumari R V. 2015. An Economic Analysis of Processing of Cotton Crop Produce. International Journal of Economic Plants, 2, 162-167.

Razzaq A, Saleem F, Kanwal M, Mustafa G, Yousaf S, Arshad H M I, Hameed M K, Khan M S, Joyia F A. 2019. Modern trends in plant genome editing: An inclusive review of the CRISPR/Cas9 toolbox. International Journal of Molecular Sciences, 20, 4045.

Ren Q, Zhong Z, Wang Y, You Q, Li Q, Yuan M Z, He Y, Qi C Y, Tang X, Zheng X L, Zhang Tao, Qi Yi P, Zhang Y. 2019. Bidirectional promoter-based CRISPR-Cas9 systems for plant genome editing. Frontiers in Plant Science, 10, 1173.

Rogers G M, Poore M H, Paschal J C. 2002. Feeding cotton products to cattle. Veterinary Clinics: Food Animal Practice, 18, 267-294.

Salimath S S, Romsdahl T B, Konda A R, Zhang W, Cahoon E B, Dowd M K, Wedegaertner T C, Hake K D, Chapman K D. 2021. Production of tocotrienols in seeds of cotton (Gossypium hirsutum L.) enhances oxidative stability and offers nutraceutical potential. Plant Biotechnology Journal, 19, 1268-1282.

Sun L, Alariqi M, Wang Y X, Wang Q Q, Xu Z P, Zafar M N, Yang G Q, Jia R Y, Hussain A, Chen Y L, Ding X, Zhou J W, Wang G Y, Wang F Q, Li J Y, Zou J W, Zhu X Q, Yu L, Sun Y W, Liang S J, et al. 2024. Construction of host plant insect-resistance mutant library by high-throughput CRISPR/Cas9 system and identification of a broad-spectrum insect resistance gene. Advanced Science, 11, e2306157.

Sun L, Alariqi M, Zhu Y, Li J Y, Li Z L, Wang Q, Li Ya J, Rui H P, Zhang X L, Jin S X. 2018. Red fluorescent protein (DsRed2), an ideal reporter for cotton genetic transformation and molecular breeding. The Crop Journal, 6, 366-376.

Sunilkumar G, Connell J P, Smith C W, Reddy A S, Rathore K S. 2002. Cotton α-globulin promoter: Isolation and functional characterization in transgenic cotton, Arabidopsis, and tobacco. Transgenic Research, 11, 347-359.

Swinnen G, Goossens A, Pauwels L. 2016. Lessons from domestication: Targeting cis-regulatory elements for crop improvement. Trends in Plant Sci全称, 21, 506-515.

Tang X, Ren Q R, Yang L J, Bao Y, Zhong Z H, He Y, Liu S S, Qi C Y, Liu B L, Wang Y, Sretenovic S, Zhang Y X, Zheng X L, Zhang T, Qi Y P, Zhang Y. 2019. Single transcript unit CRISPR 2.0 systems for robust Cas9 and Cas12a mediated plant genome editing. Plant Biotechnology Journal, 17, 1431-1445.

Taway K, Dachphun I, Vuttipongchaikij S, Suttangkakul A. 2023. Evaluation of cucumber UBL5 promoter as a tool for transgene expression and genome editing in plants. Transgenic Research, 32, 437-449.

Townsend J A, Wright D A, Winfrey R J, Fu F L, Maeder M L, Joung J K, Voytas D F. 2009. High-frequency modification of plant genes using engineered zinc-finger nucleases. Nature, 459, 442-445.

Wang G Y, Wang F Q, Xu Z P, Wang Y, Zhang C, Zhou Y, Hui F J, Yang X Y, Nie X H, Zhang X L, Jin S X. 2024. Precise fine-turning of GhTFL1 by base editing tools defines ideal cotton plant architecture. Genome Biology, 25, 59.

Wang G Y, Xu Z P, Wang F Q, Huang Y F, Xin Y F, Liang S J, Li B, Si H, Sun L, Wang Q Q, Ding X, Zhu X Q, Chen L, Yu L, Lindsey K, Zhang X L, Jin S X. 2022. Development of an efficient and precise adenine base editor (ABE) with expanded target range in allotetraploid cotton (Gossypium hirsutum). BMC Biology, 20, 45.

Wang Q Q, Alariqi M, Wang F Q, Li B, Ding X, Rui H P, Li Y J, Xu Z P, Qin L, Sun L, Li J Y, Zou J W, Lindsey K, Zhang X L, Jin S X. 2020. The application of a heat‐inducible CRISPR/Cas12b (C2c1) genome editing system in tetraploid cotton (G. hirsutum) plants. Plant Biotechnology Journal, 18, 2436-2443.

Wilson F D, Smith J N. 1976. Some genetic relationships between gland density and gossypol content in Gossypium hirsutum L.1. Crop Science, 16, 830-832.

Wu T J, Sun J Y, Lu L J, Wang C, Zhou S W, Chen Y L, Wang X J, Wang X L. 2024. Rapid on-site genotyping of the ovine prolific FecBB mutation using a CRISPR/Cas12a-based detection system. Journal of Integrative Agriculture, doi.org/10.1016/j.jia.2024.05.013.

Xu Y X, Wang G N, Shao L M, Wang N, She L X, Liu Y, Geng Y H, Yan G. 2023. GlandSegNet: Semantic segmentation model and area detection method for cotton leaf pigment glands. Computers and Electronics in Agriculture, 212, 108130.

Yang L, Li L, Liu H Y, Li S, Xing F, Chen L L. 2014. CRISPR-P: A web tool for synthetic single-guide RNA design of CRISPR-system in plants. Molecular Plant, 7, 1494-1496.

Yang Y, Li X R, Li C Y, Zhang H, Tuerxun Z, Hui F J, Li J, Liu Z G, Chen G, Cai D R, Chen X J, Li B. 2024. Isolation and functional characterization of a constitutive promoter in upland cotton (Gossypium hirsutum L.). International Journal of Molecular Sciences, 25, 1917.

Yang Z Q, Wang J, Huang Y M, Wang S B, Wei L L, Liu D X, Weng Y L, Xiang J H, Zhu Q, Yang Z E, Nie X H, Yu Y, Yang Z R, Yang Q Y. 2023. CottonMD: A multi-omics database for cotton biological study. Nucleic Acids Research, 51, D1446-D1456.

Yu L, Li Z S, Ding X, Alariqi M, Zhang C J, Zhu X Q, Fan S L, Zhu L F, Zhang X L, Jin S X. 2023. Developing an efficient CRISPR-dCas9-TV-derived transcriptional activation system to create three novel cotton germplasm materials. Plant Communications, 4, 100600.

Zetsche B, Gootenberg J S, Abudayyeh O O, Slaymaker I M, Makarova K S, Essletzbichler P, Volz S E, Joung J, Oost J V D, Regev A, Koonin E V, Zhang F. 2015. Cas12a is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163, 759-771.

Zhang Y, Ren Q, Tang X, Liu S S, Malzahn A A, Zhou J P, Wang J H, Yin D S, Pan C T, Yuan M Z, Huang L, Yang H, Zhao Y X, Fang Q, Zheng X L, Tian L, Cheng Y H, Le Y, McCoy B, Franklin Let al. 2021. Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems. Nature Communications, 12, 1944.

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

Zhou J P, Liu G Q, Zhao Y X, Zhang R, Tang Xu, Li L, Jia X Y, Guo Y C, Wu Y C, Han Y S, Bao Y, He Y, Han Q Q, Yang H, Zheng X L, Qi Y P, Zhang T, Zhang Y. 2023. An efficient CRISPR-Cas12a promoter editing system for crop improvement. Nature Plants, 9, 588-604.

Zhu X Q, Xu Z P, Wang G Y, Cong Y L, Yu L, Jia R Y, Qin Y, Zhang G Y, Li B, Yuan D J, Tu L L, Yang X Y, Lindsey K, Zhang X L, Jin S X. 2023. Single-cell resolution analysis reveals the preparation for reprogramming the fate of stem cell niche in cotton lateral meristem. Genome Biology, 24, 194.

Zubair M F, Ibrahim O S, Atolani O, Hamid A A. 2021. Chemical composition and nutritional characterization of cotton seed as potential feed supplement. Journal of the Turkish Chemical Society Section A: Chemistry, 8, 977-982.

 

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