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.

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 wheatFADgene family (TaFADs).Results: 68FADgenes were identified from the wheat genome. Based on the phylogenetic analysis, wheatFADsclustered into five subfamilies, includingFAB2,FAD2/FAD6,FAD4,DES/SLD, andFAD3/FAD7/FAD8. TheTaFADswere 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 theTaFADs promoters indicated the role of these genes in plant development and responses to environmental stresses. Likewise, 72 SSRs and 91 miRNAs in 36 and 47TaFADs have been identified. According to RNA-seq data analysis, the highest expression in all developmental stages and tissues was related toTaFAB2.5,TaFAB2.12,TaFAB2.15,TaFAB2.17,TaFAB2.20,TaFAD2.1,TaFAD2.6, andTaFAD2.8genes while the highest expression in response to temperature stress was related toTaFAD2.6,TaFAD2.8,TaFAB2.15,TaFAB2.17, andTaFAB2.20. 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.

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.

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.

Calcium (Ca²⁺) serves as a crucial intracellular messenger in plant signaling, particularly during stress responses. Precise regulation of calcium levels by transporters such as calcium/cation (CaCA) antiporters is essential for its effective function. However, the evolutionary dynamics and stress-related roles of the CaCA superfamily remain underexplored in key Brassicaceae crops. This study aimed to address this gap by investigating the hypothesis that CaCA genes in Brassica napus, B. rapa, and B. oleracea have undergone distinct evolutionary trajectories influencing their roles in abiotic stress responses, using Arabidopsis thaliana for comparison. Using Hidden Markov Model (HMM) profiling, 93 CaCA genes were identified across these species. These genes were categorized into four phylogenetic clades: CAX, CCX, NCL, and MHX. Comprehensive analyses of their coding proteins physicochemical properties, subcellular localization, conserved motifs, and gene structures were performed. Codon usage bias (CUB) analysis showed CaCA genes have low codon bias and CUB indices indicated a complex interplay between mutational and selective pressures, highlighting the influence of natural selection and mutational biases in shaping these genes. Collinearity and duplication analyses highlighted the evolutionary dynamics of the CaCA gene family, with several segmental and whole-genome duplication (WGD) events contributing to their expansion. Notably, duplicated genes underwent negative selection pressure, which removed harmful mutations, resulting in slower evolution and maintaining the functional stability of CaCA genes throughout their evolutionary history. Analysis of cis-regulatory elements (CREs) revealed their responsiveness to hormones and stresses, suggesting a potential role in plant environmental adaptation. Expression profiling of CaCA genes under abiotic stresses (dehydration, salinity, cold, and ABA) in B. napus was performed using publicly available RNA-seq datasets and analyzed with standard bioinformatics tools. Based on the results of expression analysis, key CaCA genes, such as BnCAX3, BnCAX16, BnCC2, BnCCX9, BnCAX5, BnCAX12, BnCAX13, and BnMHX1, which are differentially expressed and potentially crucial for stress tolerance. This comprehensive study elucidates the evolutionary architecture of the CaCA gene family in Brassicaceae and identifies key BnCaCA genes potentially crucial for abiotic stress tolerance, thus offering a foundation for future functional studies aimed at improving crop resilience.