Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9

CRISPR/Cas （clustered regularly interspaced short palindromic repeats/CR1SPR-associated proteins） is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic To plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67 %. Stable knockout transformants for maize phytoene synthase gene （PSYI） were obtained. Mutations occurred in germ ceils can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.

Wheat genomic study for genetic improvement of traits in China

Bread wheat(Triticum aestivum L.)is a major crop that feeds 40%of the world’s population.Over the past several decades,advances in genomics have led to tremendous achievements in understanding the origin and domestication of wheat,and the genetic basis of agronomically important traits,which promote the breeding of elite varieties.In this review,we focus on progress that has been made in genomic research and genetic improvement of traits such as grain yield,end-use traits,flowering regulation,nutrient use efficiency,and biotic and abiotic stress responses,and various breeding strategies that contributed mainly by Chinese scientists.Functional genomic research in wheat is entering a new era with the availability of multiple reference wheat genome assemblies and the development of cutting-edge technologies such as precise genome editing tools,highthroughput phenotyping platforms,sequencing-based cloning strategies,high-efficiency genetic transformation systems,and speed-breeding facilities.These insights will further extend our understanding of the molecular mechanisms and regulatory networks underlying agronomic traits and facilitate the breeding process,ultimately contributing to more sustainable agriculture in China and throughout the world.

Defective Kernel 39 encodes a PPR protein required for seed development in maize

RNA editing is a posttranscriptional process that is important in mitochondria and plastids of higher plants. All RNA editing-specific trans-factors reported so far belong to PLS-class of pentatricopeptide repeat(PPR)proteins. Here, we report the map-based cloning and molecular characterization of a defective kernel mutant dek39 in maize. Loss of Dek39 function leads to delayed embryogenesis and endosperm development, reduced kernel size, and seedling lethality. Dek39 encodes an E subclass PPR protein that targets to both mitochondria and chloroplasts, and is involved in RNA editing in mitochondrial NADH dehydrogenase3(nad3) at nad3-247 and nad3-275. C-to-U editing of nad3-275 is not conserved and even lost in Arabidopsis, consistent with the idea that no close DEK39 homologs are present in Arabidopsis. However, the amino acids generated by editing nad3-247 and nad3-275 are highly conserved in many other plant species, and the reductions of editing at these two sites decrease the activity of mitochondria NADH dehydrogenase complex I,indicating that the alteration of amino acid sequence is necessary for Nad3 function. Our results indicate that Dek39 encodes an E sub-class PPR protein that is involved in RNA editing of multiple sites and is necessary for seed development of maize.