Efficient BoPDS Gene Editing in Cabbage by the CRISPR/Cas9 System

Ma, Cunfa; Liu, Mengci; Li, Qinfei; Sheng, Jun; Ren, Xuesong; Song, Houyan

doi:10.1016/j.hpj.2019.04.001

Cited by 51 publications

(15 citation statements)

References 37 publications

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“…Traditional genetic crossing/breeding approaches to obtain higher order mutants, however, are both time-consuming and complicated by genetic linkage. Compared to traditional breeding, CRISPR-Cas9 gene editing has been recently used to study redundant gene families in several plant species such as rice, wheat, cabbage, tomato and Arabidopsis [21,[26][27][28][29]. The research reported here, however, represents the first application of CRISPR/Cas9 to understand GTs families in Arabidopsis.…”

Section: Discussionmentioning

confidence: 99%

Elucidating the roles of three β-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis

2020

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Background: Arabinogalactan-proteins (AGPs) are one of the most complex protein families in the plant kingdom and are present in the cell walls of all land plants. AGPs are implicated in diverse biological processes such as plant growth, development, reproduction, and stress responses. AGPs are extensively glycosylated by the addition of type II arabinogalactan (AG) polysaccharides to hydroxyproline residues in their protein cores. Glucuronic acid (GlcA) is the only negatively charged sugar added to AGPs and the functions of GlcA residues on AGPs remain to be elucidated. Results: Three members of the CAZy GT14 family (GLCAT14A-At5g39990, GLCAT14B-At5g15050, and GLCAT14C-At2g37585), which are responsible for transferring glucuronic acid (GlcA) to AGPs, were functionally characterized using a CRISPR/Cas9 gene editing approach in Arabidopsis. RNA seq and qRT-PCR data showed all three GLCAT genes were broadly expressed in different plant tissues, with GLCAT14A and GLCAT14B showing particularly high expression in the micropylar endosperm. Biochemical analysis of the AGPs from knockout mutants of various glcat single, double, and triple mutants revealed that double and triple mutants generally had small increases of Ara and Gal and concomitant reductions of GlcA, particularly in the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants. Moreover, AGPs isolated from all the glcat mutants displayed significant reductions in calcium binding compared to WT. Further phenotypic analyses found that the glcat14a glcat14b and glcat14a glcat14b glcat14c mutants exhibited significant delays in seed germination, reductions in root hair length, reductions in trichome branching, and accumulation of defective pollen grains. Additionally, both glcat14b glcat14c and glcat14a glcat14b glcat14c displayed significantly shorter siliques and reduced seed set. Finally, all higher-order mutants exhibited significant reductions in adherent seed coat mucilage.

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Section: Discussionmentioning

confidence: 99%

Elucidating the roles of three β-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis

2020

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“…With continuous selection on Km, the frequency of mutations detected by the restriction enzyme site loss method varied between 47 and 88%. Similarly, high efficiencies were detected with Agrobacterium-mediated gene editing with stable transgene integration for the ALS gene in potato [6,7,14] or for PDS in other plant species: tomato, poplar, apple, rice, watermelon, cassava, grape, banana, coffee, pear, chicory, strawberry and cabbage [16,[19][20][21][22][23][24][25][26][27][28][29][30].…”

Section: Discussionmentioning

confidence: 95%

“…However, this phenomenon is not without examples, as large deletions were identified in the Gn1a gene of rice and the ALARP gene of cotton [31,32]. Deletions larger than 100 bp are generally found only after mutagenesis with double or multiple sgRNA constructs [30,[33][34][35][36][37][38]. In potato, a deletion larger than 100 bp was detected in one out of nine mutants obtained in the Pi transportrelated PHO1 gene [39].…”

Section: Discussionmentioning

confidence: 99%

Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation

et al. 2020

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Background: Gene editing using the CRISPR/Cas9 system has become a routinely applied method in several plant species. The most convenient gene delivery system is Agrobacterium-mediated gene transfer with antibiotic selection and stable genomic integration of transgenes, including Cas9. For elimination of transgenes in the segregating progeny, selfing is applied in many plant species. This approach, however, cannot be widely employed in potato because most of the commercial potato cultivars are self-incompatible. Results: In this study, the efficiency of a transient Cas9 expression system with positive/negative selection based on codA-nptII fusion was tested. The PHYTOENE DESATURASE (PDS) gene involved in carotenoid biosynthesis was targeted. A new vector designated PROGED::gPDS carrying only the right border of T-DNA was constructed. Using only the positive selection function of PROGED::gPDS and the restriction enzyme site loss method in PCR of genomic DNA after digestion with the appropriate restriction enzyme, it was demonstrated that the new vector is as efficient in gene editing as a traditional binary vector with right-and left-border sequences. Nevertheless, 2 weeks of positive selection followed by negative selection did not result in the isolation of PDS mutants. In contrast, we found that with 3-day positive selection, PDS mutants appear in the regenerating population with a minimum frequency of 2-10%. Interestingly, while large deletions (> 100 bp) were generated by continuous positive selection, the 3-day selection resulted in deletions and substitutions of only a few bp. Two albinos and three chimaeras with white and green leaf areas were found among the PDS mutants, while all the other PDS mutant plants were green. Based on DNA sequence analysis some of the green plants were also chimaeras. Upon vegetative propagation from stem segments in vitro, the phenotype of the plants obtained even by positive selection did not change, suggesting that the expression of Cas9 and gPDS is silenced or that the DNA repair system is highly active during the vegetative growth phase in potato. Conclusions: Gene-edited plants can be obtained from potatoes by Agrobacterium-mediated transformation with 3-day antibiotic selection with a frequency high enough to identify the mutants in the regenerating plant population using PCR.

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“…var) using customized TALEN based nuclease constructed using a method "unit assembly" specially targeted the endogenous FRIGIDA gene in Brasica oreracea L. var modified the targeted site with deletion and this protocol is proven to bring genetic modification by sitedirected mutagenesis (82). Two other research works that have been reported on a targeted mutation of the cabbage genome using the CRISPR/Cas9 system (83,84). One report has been done on BoPDS gene knockout and produced albino cabbage ( Brassica oleracea ) shoot and the mutation rate was 1.14% (83).…”

Section: Cabbage (Brasica Oreracea) Improvement Through Site-directed Mutagenesismentioning

confidence: 99%

“…Two other research works that have been reported on a targeted mutation of the cabbage genome using the CRISPR/Cas9 system (83,84). One report has been done on BoPDS gene knockout and produced albino cabbage ( Brassica oleracea ) shoot and the mutation rate was 1.14% (83). By another work report Knockout of Bnlpat2 and Bnlpat5 genes have been done with mutation rate ranging 17 to 68% to improve the seed starch content and increased oil bodies of the matured seed of cabbage (84).…”

Section: Cabbage (Brasica Oreracea) Improvement Through Site-directed Mutagenesismentioning

confidence: 99%

The potential applications of site-directed mutagenesis for crop improvement: a review

Bezie

Tilahun

Atnaf

2020

J. Crop Sci. Biotechnol.

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The search for technologies for crop improvement has been a continuous practice to address the food insecurity to the growing human population with an ever decreasing arable land and dynamic climate change around the world. Considering the potential technologies for crop improvement could close the rooms of poverty in developing countries in particular and around the globe at large. This review aimed to assess the site-directed mutation creation methods and to show the potential tools for future crop improvement programs. Site-directed mutagenesis was found to be an efficient process to create targeted mutation on cereal crops, horticultural crops, oilseed crops, and others. Agronomic traits such as yield, quality, and .

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Efficient BoPDS Gene Editing in Cabbage by the CRISPR/Cas9 System

Cited by 51 publications

References 37 publications

Elucidating the roles of three β-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis

Elucidating the roles of three β-glucuronosyltransferases (GLCATs) acting on arabinogalactan-proteins using a CRISPR-Cas9 multiplexing approach in Arabidopsis

Generation of transgene-free PDS mutants in potato by Agrobacterium-mediated transformation

The potential applications of site-directed mutagenesis for crop improvement: a review

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