Efficient genome editing in grapevine using CRISPR/LbCas12a system.

Clustered regularly interspaced short palindromic repeats (CRISPR) /Cas12a system, also known as CRISPR/Cpf1, has been successfully harnessed for genome engineering in many plants, but not in grapevine yet. Here we developed and demonstrated the efficacy of CRISPR/Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) in inducing targeted mutagenesis by targeting the tonoplastic monosaccharide transporter1 (TMT1) and dihydroflavonol-4-reductase 1 (DFR1) genes in 41B cells. Knockout of DFR1 gene altered flavonoid accumulation in dfr1 mutant cells. Heat treatment (34℃) improved the editing efficiencies of CRISPR/LbCas12a system, and the editing efficiencies of TMT1-crRNA1 and TMT1-crRNA2 increased from 35.3% to 44.6% and 29.9% to 37.3% after heat treatment, respectively. Moreover, the sequences of crRNAs were found to be predominant factor affecting editing efficiencies irrespective of the positions within the crRNA array designed for multiplex genome editing. In addition, genome editing with truncated crRNAs (trucrRNAs) showed that trucrRNAs with 20 nt guide sequences were as effective as original crRNAs with 24 nt guides in generating targeted mutagenesis, whereas trucrRNAs with shorter regions of target complementarity ≤ 18 nt in length may not induce detectable mutations in 41B cells. All these results provide evidence for further applications of CRISPR/LbCas12a system in grapevine as a powerful tool for genome engineering.

Gene body demethylation increases expression and is associated with self-pruning during grape genome duplication.

A colchicine-induced autotetraploid grapevine exhibiting potentially valuable agronomic traits for grape production and breeding, including self-pruning, was identified. This study investigated DNA methylation variation and its role in gene expression during self-pruning in the autotetraploid grapevine. We used RNA-Seq to estimate differentially expressed genes between diploid and autotetraploid grapevine shoot tips. The genes showing increases in the autotetraploid were mainly related to stress response pathways, whereas those showing decreases in the autotetraploid were related to biological metabolism and biosynthesis. Whole-genome bisulfite sequencing was performed to produce single-base methylomes for the diploid and autotetraploid grapevines. Comparison between the methylomes revealed that they were conserved in CG and CHG contexts. In the autotetraploid grapevine, hypodifferentially methylated regions (DMRs) and hyper-DMRs in the gene body increased or decreased gene expression, respectively. Our results indicated that a hypo-DMR in the ACO1 gene body increased its expression and might promote self-pruning. This study reports that hypo-DMRs in the gene body increase gene expression in plants and reveals the mechanism underlying the changes in the modifications affecting gene expression during genome duplication. Overall, our results provide valuable information for understanding the relationships between DNA methylation, gene expression, and autotetraploid breeding in grape.

Recovery of the non-functional EGFP-assisted identification of mutants generated by CRISPR/Cas9.

KEY MESSAGE: The recovery of non-functional-enhanced green fluorescence protein can be used as indicator to facilitate the identification of mutants generated by CRISPR/Cas9. The CRISPR/Cas9 system is a powerful tool for genome editing and it has been employed to knock out genes of interest in multiple plant species. Identification of desired mutants from regenerated plants is necessary prior to functional study. Current screening methods work based on the purification of genomic DNA and it would be laborious and time consuming using these methods to screen mutants from a large population of seedlings. Here, we developed the non-functional enhanced green fluorescence protein (nEGFP) reporter gene by inserting a single guide RNA (sgRNA) and the protospacer adjacent motif in the 5' coding region of EGFP, and the activity of nEGFP could be recovered after successful targeted editing. Using the nEGFP as the reporter gene in Nicotiana tabacum, we found that over 94% of the plants exhibiting EGFP fluorescence were confirmed to be desired mutants. The use of this nEGFP reporter construct had limited negative effect on editing efficiency, and the expression of Cas9 and sgRNA was not affected. Moreover, this method was also applied in grape by targeting the phytoene desaturase gene (PDS), and the grape cells with EGFP signal were revealed to contain targeted mutations in VvPDS. Our results show that the nEGFP gene can be used as reporter to help screen mutants according to the recovered EGFP fluorescence during the application of CRISPR/Cas9 in plants.