Nucleoporin 50 proteins affect longevity and salinity stress tolerance in seeds.

Nucleoporin 50 (Nup50) is an evolutionarily conserved protein that is a constituent of the nuclear pore complex (NPC); however, its physiological role in plants is unclear. Arabidopsis has two Nup50 proteins, Nup50a and Nup50b, which are highly expressed in developing seeds. Green fluoresceent protein (GFP)-fused Nup50a and Nup50b are localized exclusively in the nucleopolasm, implying an additional function beyond the NPC in the nuclear envelope. To investigate the function of Nup50s, we employed the CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] system to generate a nup50a nup50b double mutant, which exhibited premature translation termination of both Nup50 proteins. While the mutant showed no significant abnormal phenotype during vegetative growth, the nup50a nup50b seeds had an abnormal shape compared with the wild type. Comparative transcriptomics using immature seeds revealed that Nup50s regulate the expression of various genes, including cell wall-related genes. The nup50a nup50b seeds exhibited reduced seed longevity and salinity stress tolerance. Tetrazolium uptake and mucilage release assays implied that the nup50a nup50b seeds had greater water permeability than the wild type. Taken together, our results imply that Nup50s play a critical role in seed formation by regulating gene expression.

Simple-to-use CRISPR-SpCas9/SaCas9/AsCas12a vector series for genome editing in Saccharomyces cerevisiae.

Genome editing using the CRISPR/Cas system has been implemented for various organisms and becomes increasingly popular even in the genetically tractable budding yeast Saccharomyces cerevisiae. Because each CRISPR/Cas system recognizes only the sequences flanked by its unique protospacer adjacent motif (PAM), a certain single system often fails to target a region of interest due to the lack of PAM, thus necessitating the use of another system with a different PAM. Three CRISPR/Cas systems with distinct PAMs, namely SpCas9, SaCas9, and AsCas12a, have been successfully used in yeast genome editing. Their combined use should expand the repertoire of editable targets. However, currently available plasmids for these systems were individually developed under different design principles, thus hampering their seamless use in the practice of genome editing. Here, we report a series of Golden Gate Assembly-compatible backbone vectors designed under a unified principle to exploit the three CRISPR/Cas systems in yeast genome editing. We also created a program to assist the design of genome-editing plasmids for individual target sequences using the backbone vectors. Genome editing with these plasmids demonstrated practically sufficient efficiency in the insertion of gene fragments to essential genes (median 52.1%), the complete deletion of an open reading frame (median 78.9%), and the introduction of single amino acid substitutions (median 79.2%). The backbone vectors with the program would provide a versatile toolbox to facilitate the seamless use of SpCas9, SaCas9, and AsCas12a in various types of genome manipulation, especially those that are difficult to perform with conventional techniques in yeast genetics.