Cationic lipid nanoparticle-mediated delivery of a Cas9/crRNA ribonucleoprotein complex for transgene-free editing of the citrus plant genome.

KEY MESSAGE: A lipofectamine-mediated transfection protocol for DNA-free genome editing of citrus protoplast cells using a Cas9/gRNA ribonucleoprotein (RNP) complex resulted in the production of transgene free genome edited citrus.

Aeluropus littoralis stress-associated protein promotes water deficit resilience in engineered durum wheat.

Global climate change-related water deficit negatively affect the growth, development and yield performance of multiple cereal crops, including durum wheat. Therefore, the improvement of water-deficit stress tolerance in durum wheat varieties in arid and semiarid areas has become imperative for food security. Herein, we evaluated the water deficiency resilience potential of two marker-free transgenic durum wheat lines (AlSAP-lines: K9.3 and K21.3) under well-watered and water-deficit stress conditions at both physiological and agronomic levels. These two lines overexpressed the AlSAP gene, isolated from the halophyte grass Aeluropus littoralis, encoding a stress-associated zinc finger protein containing the A20/AN1 domains. Under well-watered conditions, the wild-type (WT) and both AlSAP-lines displayed comparable performance concerning all the evaluated parameters. Ectopic transgene expression exerted no adverse effects on growth and yield performance of the durum wheat plants. Under water-deficit conditions, no significant differences in the plant height, leaf number, spike length, and spikelet number were observed between AlSAP-lines and WT plants. However, compared to WT, the AlSAP-lines exhibited greater dry matter production, greater flag leaf area, improved net photosynthetic rate, stomatal conductance, and water use efficiency. Notably, the AlSAP-lines displayed 25 % higher grain yield (GY) than the WT plants under water-deficit conditions. The RT-qPCR-based selected stress-related gene (TdDREB1, TdLEA, TdAPX1, and TdBlt101-2) expression analyses indicated stress-related genes enhancement in AlSAP-durum wheat plants under both well-watered and water-deficit conditions, potentially related to the water-deficit resilience. Collectively, our findings support that the ectopic AlSAP expression in durum wheat lines enhances water-deficit resilience ability, thereby potentially compensate for the GY loss in arid and semi-arid regions.

Development of a marker-free engineered durum wheat overexpressing Lobularia maritima GASA1 with improved drought tolerance.

Due to their fixed lifestyle, plants must adapt to abiotic or biotic stresses by orchestrating various responses, including protective and growth control measures. Growth arrest is provoked upon abiotic stress and can impair plant production. Members of the plant-specific GASA (gibberellic acid-stimulated Arabidopsis) gene family play crucial roles in phytohormone responses, abiotic and biotic stresses, and plant growth. Here, we recognized and examined the LmGASA1 gene from the halophyte plant Lobularia maritima and developed marker-free engineered durum wheat plants overexpressing the gene. The LmGASA1 transcript profile revealed that it's induced by stressful events as well as by phytohormones including GA3, MeJA, and ABA, suggesting that the LmGASA1 gene may contribute to these stress and hormone signal transduction pathways. Transient expression of GFP-LmGASA1 fusion in onion epidermal cells indicated that LmGASA1 is localized to the cell membrane. Further analysis showed that overexpression of LmGASA1 in durum wheat plants enhanced tolerance to drought stress compared with that in non-transgenic (NT) plants, imposing no yield penalty and enabling seed production even following drought stress at the vegetative stage. Altogether, our data indicate that LmGASA1 regulates both the scavenging capacity of the antioxidant enzymatic system and the activation of at least six stress-related genes that function as positive regulators of drought stress tolerance. LmGASA1 appears to be a novel gene useful for further functional analysis and potential engineering for drought stress tolerance in crops.

CRISPR/Cas9 Ribonucleoprotein-Mediated Mutagenesis in Sporisorium reilianum.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) has become the state of the art for mutagenesis in filamentous fungi. Here, we describe a ribonucleoprotein complex (RNP)-mediated CRISPR/Cas9 for mutagenesis in Sporisorium reilianum. The efficiency of the method was tested in vitro with a cleavage assay as well as in vivo with a GFP-expressing S. reilianum strain. We applied this method to generate frameshift- and knock-out mutants in S. reilianum without a resistance marker by using an auto-replicating plasmid for selection. The RNP-mediated CRISPR/Cas9 increased the mutagenesis efficiency, can be applied for all kinds of mutations, and enables a marker-free genome editing in S. reilianum. Key features • First CRISPR/Cas9 application in S. reilianum. • Generation of S. reilianum mutants without genomic integration of resistance marker. • Allows the generation of multiple gene knockouts as well as deletion of large genomic regions.

Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker-free gene editing in Trypanosoma and Leishmania.

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite.

Efficient expression of γ-glutamyl transpeptidase in Bacillus subtilis via CRISPR/Cas9n and its immobilization.

In this study, we successfully applied the strategy of combining tandem promoters and tandem signal peptides with overexpressing signal peptidase to efficiently express and produce γ-glutamyl peptidase (GGT) enzymes (BsGGT, BaGGT, and BlGGT) from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis in Bacillus subtilis ATCC6051Δ5. In order to avoid the problem of instability caused by duplicated strong promoters, we assembled tandem promoters of different homologous genes from different species. To achieve resistance marker-free enzyme in the food industry, we first removed the replication origin and corresponding resistance marker of Escherichia coli from the expression vector. The plasmid was then transformed into the B. subtilis host, and the Kan resistance gene in the expression plasmid was directly edited and silenced using the CRISPR/Cas9n-AID base editing system. As a result, a recombinant protein expression carrier without resistance markers was constructed, and the enzyme activity of the BlGGT strain during shake flask fermentation can reach 53.65 U/mL. The recombinant BlGGT was immobilized with epoxy resin and maintained 82.8% enzyme activity after repeated use for 10 times and 87.36% enzyme activity after storage at 4 °C for 2 months. The immobilized BlGGT enzyme was used for the continuous synthesis of theanine with a conversion rate of 65.38%. These results indicated that our approach was a promising solution for improving enzyme production efficiency and achieving safe production of enzyme preparations in the food industry. KEY POINTS: • Efficient expression of recombinant proteins by a combination of dual promoter and dual signal peptide. • Construction of small vectors without resistance markers in B. subtilis using CRISPR/Cas9n-AID editing system. • The process of immobilizing BlGGT with epoxy resin was optimized.

Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit.

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis 'Hongyang' and A.eriantha 'White'). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.

DNA- and Selectable-Marker-Free Genome-Editing System Using Zygotes from Recalcitrant Maize Inbred B73.

Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involve insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, using such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes successfully developed and were regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics.