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CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry
Authors:Han  Xiaoyan  Yang  Yingzhen  Han  Xue  Ryner  John T  Ahmed  Emadeldin A H  Qi  Yiping  Zhong  Gan-yuan  Song  Guo-qing
Institution:1.Plant Biotechnology Resource and Outreach Center, Department of Horticulture, Michigan State University, East Lansing, MI, 48824, USA
;2.Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People’s Republic of China
;3.Grape Genetics Research Unit, USDA-Agricultural Research Service, Geneva, NY, 14456, USA
;4.Breeding Research Department of Fruit Trees, Ornamental and Woody Plants, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt
;5.Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, 20742, USA
;
Abstract:

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 (T0) 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.

Keywords:
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