Efficient large fragment deletion in plants:double pairs of sgRNAs are better than dual sgRNAs

Dear Editor,Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR)is a powerful and versatile gene editing system that has been extensively utilized in various animals and plants,which holds enormous potential and value for scientific research and breeding.However,single-targeted CRISPR can only induce a few base deletions,insertions,or substitution.Ideally,thesemutations result in premature termination of the protein encoded by the target gene,leading to a loss of function[1].

Diversity and redundancy of the ripening regulatory networks revealed by the fruitENCODE and the new CRISPR/Cas9 CNR and NOR mutants

Tomato is considered as the genetic model for climacteric fruits,in which three major players control the fruit ripening process:ethylene,ripening transcription factors,and DNA methylation.The fruitENCODE project has now shown that there are multiple transcriptional circuits regulating fruit ripening in different species,and H3K27me3,instead of DNA methylation,plays a conserved role in restricting these ripening pathways.In addition,the function of the core tomato ripening transcription factors is now being questioned.We have employed CRISPR/Cas9 genome editing to mutate the SBP-CNR and NAC-NOR transcription factors,both of which are considered as master regulators in the current tomato ripening model.These plants only displayed delayed or partial non-ripening phenotypes,distinct from the original mutant plants,which categorically failed to ripen,suggesting that they might be gain-of-function mutants.Besides increased DNA methylation genome-wide,the original mutants also have hyper-H3K27me3 in ripening gene loci such as ACS2,RIN,and TDR4.It is most likely that multiple genetic and epigenetic factors have contributed to their strong non-ripening phenotypes.Hence,we propose that the field should move beyond these linear and twodimensional models and embrace the fact that important biological processes such as ripening are often regulated by highly redundant network with inputs from multiple levels.

Antibody Titer Detection and Expression Characteristic Analysis of TypeⅡTomato Metacaspase Sl MC7 Protein in Fruit

SlMC7 prokaryotic expression recombinant protein was used as test materials. SlMC7 polyclonal antiserum was obtained from immunized mice by using purified SlMC7 recombinant protein from Escherichia coli. ELISA and protein dot plot methods were used to detect the antibody titer and expression characteristics and the expression difference at different stages,providing material foundation and reference basis for subsequent SlMC7 functional research in tomato fruit. The results showed that the titer of SlMC7 polyclonal antibodies was more than 1∶ 256 000 and the sensitivity of SlMC7 polyclonal antibodies was 1. 6 ng at the dilutability of 1∶ 10 000. SlMC7 protein was detected at different stages,and SlMC7 had a higher expression level in color-break fruit,indicating that SlMC7 might be relevant to the fruit ripening of tomato.