CRISPR Cas9-and Cas12a-mediated gusA editing in transgenic blueberry

doi:10.1007/s11240-021-02177-1

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	CRISPR Cas9-and Cas12a-mediated gusA editing in transgenic blueberry
	Han, Xiaoyan 2; Yang, Yingzhen ; Han, Xue ; Ryner, John T.; Ahmed, Emadeldin A. H.; Qi, Yiping 4; Zhong, Gan-Yuan ; Song, Guo-Qing
刊名	PLANT CELL TISSUE AND ORGAN CULTURE
	2022
卷号	148 期号:2 页码:217-229
关键词	Blueberry CRISPR-Cas9 CRISPR-Cas12a Gene editing GUS
ISSN号	0167-6857
DOI	10.1007/s11240-021-02177-1
文献子类	Article
英文摘要	Key Message A second round of regeneration enriched editing events and enhanced the production of edited blueberry shoots. The new protocol described facilitates high-precision breeding of blueberries using CRISPR Cas technologies. To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T-0) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies.
学科主题	Biotechnology & Applied Microbiology ; Plant Sciences
电子版国际标准刊号	1573-5044
出版地	DORDRECHT
WOS关键词	TARGETED MUTAGENESIS ; DNA ; PLANTS ; ARABIDOPSIS ; CANCER ; TISSUE ; WHEAT
WOS研究方向	Science Citation Index Expanded (SCI-EXPANDED)
语种	英语
出版者	SPRINGER
WOS记录号	WOS:000709223900001
资助机构	USDA Agricultural Research Service and Michigan State University [58-8060-6-009] ; CAS Scholarship
内容类型	期刊论文
源URL	[http://ir.ibcas.ac.cn/handle/2S10CLM1/28548]
专题	植物园
作者单位	1.Chinese Acad Sci, Beijing Bot Garden, Inst Bot, Beijing 100093, Peoples R China 2.Michigan State Univ, Dept Hort, Plant Biotechnol Resource & Outreach Ctr, E Lansing, MI 48824 USA 3.Univ Maryland, Dept Plant Sci & Landscape Architecture, College Pk, MD 20742 USA 4.Ahmed, Emadeldin A. H.] Agr Res Ctr, Breeding Res Dept Fruit Trees Ornamental & Woody, Inst Hort Res, Giza, Egypt 5.USDA Agr Res Serv, Grape Genet Res Unit, Geneva, NY 14456 USA
推荐引用方式 GB/T 7714	Han, Xiaoyan,Yang, Yingzhen,Han, Xue,et al. CRISPR Cas9-and Cas12a-mediated gusA editing in transgenic blueberry[J]. PLANT CELL TISSUE AND ORGAN CULTURE,2022,148(2):217-229.
APA	Han, Xiaoyan.,Yang, Yingzhen.,Han, Xue.,Ryner, John T..,Ahmed, Emadeldin A. H..,...&Song, Guo-Qing.(2022).CRISPR Cas9-and Cas12a-mediated gusA editing in transgenic blueberry.PLANT CELL TISSUE AND ORGAN CULTURE,148(2),217-229.
MLA	Han, Xiaoyan,et al."CRISPR Cas9-and Cas12a-mediated gusA editing in transgenic blueberry".PLANT CELL TISSUE AND ORGAN CULTURE 148.2(2022):217-229.