Multigene editing via CRISPR/Cas9 guided by a single-sgRNA seed in Arabidopsis

We report that a solo single-guide RNA （sgRNA） seed is capable of guiding Clustered Regularly Interspaced Short Palindromic Repeats （CRISPR）/CRISPR -associated 9 （CRISRP/Casg） to simultaneously edit multiple genes AtRPL10A, AtRPL10B and AtRPL10C in Arabidopsis. Our results also demonstrate that it is possible to use CRISPR/Cas9 technology to create AtRPLlo triple mutants which otherwise cannot be generated by conventional genetic crossing. Compared to other conventional multiplex CRISPR/Cas systems, a single sgRNA seed has the advantage of reducing off-target gene-editing. Such a gene editing system might be also applicable to modify other homologous genes, or even less-homologous sequences for multiple gene-editing in plants and other organisms.

Comparative WGBS identifies genes that influence non-ripe phenotype in tomato epimutant Colourless non-ripening

Whole-genome bisulfite sequencing(WGBS)allows single-base resolution and genome-wide profiling of DNA methylation in plants and animals.This technology provides a powerful tool to identify genes that are potentially controlled by dynamic changes of DNA methylation and demethylation.However,naturally occurring epimutants are rare and genes under epigenetic regulation as well as their biological relevances are often difficult to define.In tomato,fruit development and ripening are a complex process that involves epigenetic control.We have taken the advantage of the tomato epimutant Colourless non-ripening(Cnr)and performed comparative mining of the WGBS datasets for the Cnr and Sl CMT3-silenced Cnr fruits.We compared DNA methylation profiles for the promoter sequences of approximately 5,000 bp immediately upstream of the coding region of a list of20 genes.Differentially methylated regions were found for some of these genes.Virus-induced gene silencing(VIGS)of differentially methylated gene Sl DET1 or Sl PDS resulted in unusual brown pigmentation in Cnr fruits.These results suggest that comparative WGBS coupled with VIGS can be used to identify genes that may contribute to the colourless unripe phenotype of fruit in the Cnr epimutant.