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

Authors: HAN, XIAOYAN - Michigan State University; Yang, Yingzhen; HAN, XUE - Michigan State University; RYNER, JOHN - Michigan State University; AHMED, EMADELDIN - Michigan State University; QI, YIPING - University Of Maryland; Zhong, Gan-Yuan; SONG, GUO-QING - Michigan State University

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/1/2021
Publication Date: 10/20/2021
Citation: Han, X., Yang, Y., Han, X., Ryner, J.T., Ahmed, E.A., Qi, Y., Zhong, G., Song, G. 2021. CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry. Frontiers in Plant Science. https://doi.org/10.1007/s11240-021-02177-1.
DOI: https://doi.org/10.1007/s11240-021-02177-1

Interpretive Summary: Recently, a new genomic technology termed as genome editing has been developed for modifying a gene in a precise manner without leaving any footprints. This offers an exciting solution to alleviating the public GMO concerns. This genome editing technology has well been demonstrated in several major field crops, but not yet in blueberry, which is one of the most popular fruits. Development of such a genome editing technology for blueberry will help accelerate the improvement of blueberry cultivars for meeting market needs. This study intended to develop a genome editing tool for blueberry. We evaluated genome editing efficiencies of four editing vectors involving two editing systems, termed as CRISPR-Cas9 and CRISPR-Cas12a. We chose to target (disrupt) a marker gene, beta-glucuronidase gene (gusA), in transgenic blueberry callus for demonstrating editing events. The marker gene gusA was previously introduced into blueberry cultivars and the changes of the marker gene expression can effectively be monitored through GUS staining. When the marker gene is disrupted (e.g. by editing) in a certain part of callus tissue, its gene expression and GUS staining will be compromised in that part of tissue. In our study, we found that some small proportions of the calli transformed with the four vectors had non-GUS stained sectors. Through DNA sequencing, we could confirmed that the callus transformed with one of the vectors truly had editing events. The results demonstrated that a gene could be successful edited in blueberry background, although the editing frequency was relatively low, and that significant optimization of various editing systems and components is needed for developing an efficient genome editing technology in blueberries.

Technical Abstract: Highbush blueberry cultivars (Vaccinium corymbosum L.) are typically interspecific hybrids of tetraploid and highly heterozygous. To develop an effective genome editing tool for high-precision blueberry breeding, we evaluated CRISPR-Cas9 and CRISPR-Cas12a for their editing efficiencies of a marker gene, beta-glucuronidase gene (gusA), which was previously introduced into two stable transgenic blueberry cultivars. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by the Cauliflower mosaic virus 35S promoter or the Arabidopsis ubiquitin10 promoter (AtUbi). The Cas9 vectors had two guide RNAs, one driven by Arabidopsis U6 promoter (AtU6) and the other by Arabidopsis U3 promoter (AtU3), and the Cas12a vectors had two guide RNAs in tandem driven by the AtU6 promoter. These four vectors were respectively transformed into gusA-containing blueberry leaf explants using Agrobacterium tumefaciens-mediated transformation and hygromycin selection. Gus staining showed that some small proportions of the calli transformed with the four vectors had non-GUS stained sectors. However, all individual shoots derived from the calli showed blueberry staining. We further amplified and sequenced the GUS amplicons spanning the target site in blueberry tissues of 50 samples, including 8 samples of calli with tiny shoots, 18 samples of pooled shoots, and 24 samples of individual shoots. Consistent with our histochemical GUS staining, we found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. To our surprise, little convincing editing evidence was found from the calli transformed with the AtUbi-Cas9 and the two Cas12a vectors. These results demonstrated that a gene could be successful edited in blueberry background, although the editing frequency was relatively low, and that significant optimization of various editing systems and components is needed for efficient genome editing in blueberries.