A Method to Reduce off-Targets in CRISPR/Cas9 System in Plants.

One of the strategies to reduce the off-target mutations in CRISPR/Cas9 system is to use the temperature-independent gene transformation method. Mesoporous silica nanoparticles (MSNs)-gene delivery system is temperature-independent; thus, it can transfer the interesting plasmid (pDNA) to the target plant at different temperatures, including 37 °C. Due to the high activity of SpCas9 at 37 °C compared to lower temperatures, on-target mutagenesis increases at 37 °C. Therefore, we describe the synthesis of the functionalized MSNs with the particle size of less than 40 nm, binding pDNA to the MSNs, and transferring of the pDNA-MSNs into the target plants.

Analyses of Lysin-motif Receptor-like Kinase (LysM-RLK) Gene Family in Allotetraploid Brassica napus L. and Its Progenitor Species: An In Silico Study.

The LysM receptor-like kinases (LysM-RLKs) play a crucial role in plant symbiosis and response to environmental stresses. Brassica napus, B. rapa, and B. oleracea are utilized as valuable vegetables. Different biotic and abiotic stressors affect these crops, resulting in yield losses. Therefore, genome-wide analysis of the LysM-RLK gene family was conducted. From the genome of the examined species, 33 LysM-RLK have been found. The conserved domains of Brassica LysM-RLKs were divided into three groups: LYK, LYP, and LysMn. In the BrassicaLysM-RLK gene family, only segmental duplication has occurred. The Ka/Ks ratio for the duplicated pair of genes was less than one indicating that the genes' function had not changed over time. The BrassicaLysM-RLKs contain 70 cis-elements, indicating that they are involved in stress response. 39 miRNA molecules were responsible for the post-transcriptional regulation of 12 Brassica LysM-RLKs. A total of 22 SSR loci were discovered in 16 Brassica LysM-RLKs. According to RNA-seq data, the highest expression in response to biotic stresses was related to BnLYP6. According to the docking simulations, several residues in the active sites of BnLYP6 are in direct contact with the docked chitin and could be useful in future studies to develop pathogen-resistant B. napus. This research reveals comprehensive information that could lead to the identification of potential genes for Brassica species genetic manipulation.

Identification, evolution, expression, and docking studies of fatty acid desaturase genes in wheat (Triticum aestivum L.).

BACKGROUNDS: Fatty acid desaturases (FADs) introduce a double bond into the fatty acids acyl chain resulting in unsaturated fatty acids that have essential roles in plant development and response to biotic and abiotic stresses. Wheat germ oil, one of the important by-products of wheat, can be a good alternative for edible oils with clinical advantages due to the high amount of unsaturated fatty acids. Therefore, we performed a genome-wide analysis of the wheat FAD gene family (TaFADs). RESULTS: 68 FAD genes were identified from the wheat genome. Based on the phylogenetic analysis, wheat FADs clustered into five subfamilies, including FAB2, FAD2/FAD6, FAD4, DES/SLD, and FAD3/FAD7/FAD8. The TaFADs were distributed on chromosomes 2A-7B with 0 to 10 introns. The Ka/Ks ratio was less than one for most of the duplicated pair genes revealed that the function of the genes had been maintained during the evolution. Several cis-acting elements related to hormones and stresses in the TaFADs promoters indicated the role of these genes in plant development and responses to environmental stresses. Likewise, 72 SSRs and 91 miRNAs in 36 and 47 TaFADs have been identified. According to RNA-seq data analysis, the highest expression in all developmental stages and tissues was related to TaFAB2.5, TaFAB2.12, TaFAB2.15, TaFAB2.17, TaFAB2.20, TaFAD2.1, TaFAD2.6, and TaFAD2.8 genes while the highest expression in response to temperature stress was related to TaFAD2.6, TaFAD2.8, TaFAB2.15, TaFAB2.17, and TaFAB2.20. Furthermore, docking simulations revealed several residues in the active site of TaFAD2.6 and TaFAD2.8 in close contact with the docked oleic acid that could be useful in future site-directed mutagenesis studies to increase the catalytic efficiency of them and subsequently improve agronomic quality and tolerance of wheat against environmental stresses. CONCLUSIONS: This study provides comprehensive information that can lead to the detection of candidate genes for wheat genetic modification.

In Silico Analyses of Autophagy-Related Genes in Rapeseed (Brassica napus L.) under Different Abiotic Stresses and in Various Tissues.

The autophagy-related genes (ATGs) play important roles in plant growth and response to environmental stresses. Brassica napus (B. napus) is among the most important oilseed crops, but ATGs are largely unknown in this species. Therefore, a genome-wide analysis of the B. napus ATG gene family (BnATGs) was performed. One hundred and twenty-seven ATGs were determined due to the B. napus genome, which belongs to 20 main groups. Segmental duplication occurred more than the tandem duplication in BnATGs. Ka/Ks for the most duplicated pair genes were less than one, which indicated that the negative selection occurred to maintain their function during the evolution of B. napus plants. Based on the results, BnATGs are involved in various developmental processes and respond to biotic and abiotic stresses. One hundred and seven miRNA molecules are involved in the post-transcriptional regulation of 41 BnATGs. In general, 127 simple sequence repeat marker (SSR) loci were also detected in BnATGs. Based on the RNA-seq data, the highest expression in root and silique was related to BnVTI12e, while in shoot and seed, it was BnATG8p. The expression patterns of the most BnATGs were significantly up-regulated or down-regulated responding to dehydration, salinity, abscisic acid, and cold. This research provides information that can detect candidate genes for genetic manipulation in B. napus.

A novel, simple, and stable mesoporous silica nanoparticle-based gene transformation approach in Solanum lycopersicum.

In this study, a novel and stable gene transformation system was developed under control of Maize Proteinase Inhibitor (MPI) as an inducible promoter using the Mesoporous Silica Nanoparticles (MSNs). The functionalized MSNs with a proper particle size were synthesized and attached to a recombinant construct (pDNA) containing cryIAb gene under the control of MPI promoter (pPZP122:MPI:cryIAb:MSN [pDNA: MSN]) following transformation of tomato plants through injection of the pDNA: MSN complex into tomato red fruit at early ripening stage and then, putative transgenic seeds were collected. As an initial selection, gentamicin-resistant seedlings of T1 (24.24%) and T2 (61.37%) plants were identified. The transgene integration and expression were confirmed through the PCR, RT-PCR, and western blot approaches in the selected seedlings. PCR analysis showed that transformation frequency was equal to 10.71% in T1 plants. Semi-quantitative RT-PCR analysis confirmed the transcript expression of cryIAb in all the T1 and T2 PCR-positive plants. Western blot analysis confirmed the existence of CryIAb protein in the leaves of T2 putative transgenic plants. Accordingly, the results demonstrated that the transgene has more likely integrated into the tomato genome through homologous recombination. Bioassay was carried out for further assessment of the plant responses to Tuta absoluta resulting in an enhanced tolerance of the plant. In conclusion, the MSN-mediated stable transformation system under the MPI as an inducible promoter can be used as a suitable alternative for conventional genetic transformation methods due to its biodegradability, biocompatibility, cost and time-effectiveness, and positive effect on the plant defense against pathogens and pests.

Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants.

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

Studying genetic variability of pomegranate (Punica granatum L.) based on chloroplast DNA and barcode genes.

Chloroplast DNA has been used extensively to analyze plant phylogenies at different taxonomic levels because of its size, organization and sequence conservation. In the present research, two chloroplastic regions, petA­psaJ, trnC­trnD and four DNA barcodes (trnH­psbA, ITS, rbcL, matK), were used to introduce suitable regions for the assessment of genetic diversity among P. granatum L. genotypes. Analysis of psbE­petL in petA­psaJ region revealed 1,300 nucleotides with 4.29 % genetic diversity among genotypes, while trnC­petN in trnC­trnD region showed 1.8 % genetic diversity. Therefore, despite the results obtained from the study of other plants, the trnC­trnD region had a low potential for the evaluation of diversity among pomegranate genotypes. Analysis of DNA barcodes in pomegranate showed that trnH­psbA (genetic diversity 2.91 %) provides the highest intra-species variation, followed by ITS (genetic diversity 0.44 %). Eighteen genotypes from different geographical origins of Iran were used to investigate psbE­petL and trnH­psbA potential as novel barcodes to determine genetic polymorphism and characterize pomegranate genotypes. The results suggested that two regions, psbE­petL and trnH­psbA, were more suitable for determining intra-species relationships of pomegranate.