Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

Abstract

The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

Overexpression of VqWRKY31 enhances powdery mildew resistance in grapevine by promoting salicylic acid signaling and specific metabolite synthesis

“…The VqWRKY31 cDNA was cloned by PCR using the primers 2300flg -WRKY31 (forward) and 2300flg -WRKY31 (reverse) ( Supplementary Table S1 ) and then inserted into the pCambia2300 vector downstream of the CaMV35S promoter to generate CaMV35S- VqWRKY31 -3 × Flag. A. tumefaciens GV3101 harboring 35S- VqWRKY31 -3 × Flag was used for the stable transformation of grapevine via previously described methods [ 31 ]. Young vegetative shoots from transgenic plants were transferred to agar-solidified MS medium every 2 months for rapid propagation.…”

Expanding Gene-Editing Potential in Crop Improvement with Pangenomes

“…Potentially deleterious off-target activity can occur in regions of the genome that share sequence identity with the target site such as duplicated/repeated sequences [107]. Off-target effects are often undesirable and have been observed in rice (Oryza sativa) [108], grapevine (Vitis vinifera) [109], and cotton (Gossypium) [110]. Avoiding off-target effects requires detailed genomic information for the individual being modified [107].…”

From Genome Sequencing to CRISPR-Based Genome Editing for Climate-Resilient Forest Trees

“…Recent reports in animal models using long-read sequencing demonstrated that unexpected on-target damage of CRISPR was far more serious and widespread than anticipated [217,218] and revealed unforeseen CRISPR-Cas9 off-target activity [219,220]. Recently, high-throughput whole-genome resequencing has been used to evaluate off-target edits and untargeted mutations in Arabidopsis [221], rice [222,223], tomato [224], cotton [225], and grapevine [226], confirming that the off-target CRISPR-Cas9-induced mutations are rare in plants and smaller in magnitude than the variation that is generated by conventional tissue culturing or mutation breeding [227]. However, one possible limitation of the whole-genome resequencing approach for screening a large number of potential off-target sites is that it requires a reference genome.…”