Global stability and increase of crop yield has been affected negatively in recent times due to the frequent incidents of temperature and rainfall, variations leading to heat, cold, drought and salinity stress (Abdulwahid et al., 2026) . These stressors occur as shifts in pest and disease development, ion toxicity, canopy temperature, water balance, oxidative damage and several other forms. Harnessing the precision of genome editing tools like CRISPR-Cas system and other advanced tools can be used to incorporate defined alleles in elite backgrounds, which can then be evaluated in controlled and field condition, reducing the long-time frame usually required for traditional introgression programs (Azameti and Dauda, 2021; Kalaitzandonakes et al., 2023) . There is a pressing need to translate cutting-edge biotechnological advancements into actionable solutions, ensuring the timely development of climate-resilient crops. This Research Topic was framed around translational genomics and requested research work that links stress-gene identification to actionable edits. Its intended scope includes the evaluation of genome editing platforms, optimization of protocols and delivery, and validation under conditions aligned with climate risk. The Research Topic also explicitly includes environmental and regulatory considerations and emphasizes collaborative approaches and field trials that test edited lines in production settings. 2 Highlights of the contributions 2.1 Targets and edit types for stress response and adaptation In the review article, Chavhan et al. provided a comprehensive synthesis on the application of gene editing for climate-resilient crops using CRISPR/Cas9, base editing, and prime editing. It elaborates when targeted mutagenesis, direct base conversion without double-strand breaks, or templated edits are suited to trait development. They discuss stress-response regulators and ion

Homologous recombination (HR) corrects a mutational sequence causing a genetic disease by replacing it with the normal sequence to restore a healthy state in humans. A targeted genomic breakage, such as that induced by CRISPR-Cas9, can trigger a copy-paste-type HR event; however, CRISPR-Cas9 more frequently induces imprecise non-homologous end-joining events, leading to one-step multiple knockout products for paralogous genes or homologous alleles, which can be considered a unique advantage. We have established a precision strategy for crossover-type HR-based gene editing, primed by intra-cellular circular donor cleavage (InCDC). The InCDC technique generates targeted duplication of the circular donor plasmid at the target locus in human cells, forming a doublet configuration comprising the donor DNA with the designed sequence and the target DNA with the original sequence, with much higher efficiency than conventional donor linearization techniques. This doublet form leads to the singlet form, resulting in retention of the designed allele. We found that the safety distance within the designed circular donor plasmid and its intra-cellular cleavage was particularly critical to protect a designed sequence from enzymatic exclusion, and we propose that InCDC technology enables precision genome editing, such as the replacement of a genetic disease-causing allele with the correctly designed allele.

Red leaf lettuce (Lactuca sativa L. cv. ‘Red Fire’) is a preferred crop in plant factories with artificial light (PFALs) due to its short cultivation cycle and high anthocyanin content, which increases both its nutritional value and visual appeal. However, anthocyanins strongly influence leaf coloration and antioxidant profiles, and their levels are highly responsive to the light environment. Therefore, targeted editing of flavonoid biosynthesis may provide a breeding strategy to diversify pigment composition and associated functional traits under PFAL conditions. In this study, we used CRISPR/Cas9 to knock out DFR (dihydroflavonol 4-reductase), a key enzyme in the anthocyanin pathway. Genome-edited lines were generated via a dual-guide RNA system, resulting in a successfully edited red leaf genotype. The DFR-knockout lines displayed a complete loss of red pigmentation and a visibly distinct green phenotype. Metabolite profiling revealed a significant decrease in anthocyanin levels, accompanied by an increase in total flavonoid levels in some lines. Growth traits, including shoot dry weight and leaf number, were not significantly affected, suggesting that DFR knockout does not compromise growth under PFAL conditions. These findings highlight DFR as a promising target for creating pigment-altered lettuce lines for controlled-environment cultivation, including PFAL systems.

BackgroundGenetic variations in tumor suppressor miRNAs and the 3′UTR of their target genes influence tumor biology and breast cancer (BC) risk.ObjectiveThis study investigated genetic variations in tumor suppressor miRNAs (hsa-let-7c, hsa-miR-34a, hsa-miR-145a) and their target genes (KRAS, IGFBP6, IGF1R), and their functional significance in BC patients.MethodsThe miRNA encoding regions and 3′UTRs of the selected target genes were sequenced in 208 BC patients. Functional analyses were performed using luciferase assay, RT-PCR, IHC, and Western blotting. RNAfold, TNM plot, Kaplan-Meier Plotter, and ROC Plotter were used for structural predictions, survival, and therapy response analysis.ResultsTwo variants, rs712 and rs9266, were found in the 3′UTR of KRAS. Luciferase assay confirmed that rs9266 disrupts the binding of hsa-let-7c and hsa-miR-181c, leading to increased KRAS expression. KRAS expression was highest in heterozygous, followed by homozygous mutant, and lowest in wild-type genotypes. Higher hsa-let-7c and hsa-miR-181c expression correlated with better survival. ROC analysis identified KRAS as a potential predictive biomarker for chemotherapy response.ConclusionVariants rs712 and rs9266 in the KRAS 3′UTR impair miRNA binding, enhancing KRAS expression and tumorigenesis, while elevated hsa-let-7c and hsa-miR-181c levels predict favourable survival outcomes in BC patients.

Two major processes are important for genome editing in plants: transformation by stable transfection, in which nucleic acids encoding genome-editing enzymes are introduced into plant cells and the regeneration of plant individuals from cells harboring mutations by genome-editing enzymes. The efficiency of transformation and regeneration by tissue culture varies across plant species, and is low in some practical crop species. In planta methods have been developed to exclude the need for tissue culture. However, few reports are available on methods that do not require stable transfection. Therefore, this study aimed to develop a new protocol for delivery genome editing tools that does not require transformation or tissue culture, by combining the in planta method with transient genome editing tools instead of stable transfection. Cas9, guide RNAs, and developmental regulators, which are factors involved in mitotic tissue induction, were transiently expressed by agroinfiltration of the stem tissue cut surfaces of tomatoes. New chimeric mutants, containing a mixture of cells with mutations introduced at or near the target sequence, were obtained. After examining conditions such as the concentration of Agrobacterium used for infection and post-infection treatment, we succeeded in obtaining chimeric mutants with an efficiency of 11.7%. In addition, most of the observed mutations were single base substitutions. These results indicate that the in planta method with transient expression of genome editing tools and induction of meristematic tissue can be used to introduce genome-edited mutations in tomatoes.

Our past, present, and future are governed by the grand design of the genetic blueprint, which was mapped more than twodecades back. While the eloquent design of our genome results from millions of years of evolution and has reached a near-perfect stage, unwanted flaws and mistakes in individual genomes can make their life miserable and worth intervention. The advent of technologies to manipulate the grand design and make it congenial for the individual has given new hope. Here, we discuss how gene editing technologies have progressed and how some of the technologies have become indispensable gears in precision medicine.

Pigeonpea (Cajanus cajan (L.) Millsp.; 2n = 2× = 22) is a drought-tolerant perennial grain legume commonly cultivated in India’s rain-fed and dry land zones, and it is a remarkable natural source of minerals and protein worldwide. Despite decades of research, the constraints associated with tissue culture continue to hinder the genetic improvement of the crop, including the explant’s inability to produce embryogenic calli, direct shoot formation, limited regeneration potential, and a lack of an effective transformation system. Moreover, traditional or molecular breeding approaches for crop improvement is time, resource, and labour-intensive. CRISPR/Cas9-mediated approach for targeted trait improvement has emerged as a robust technology for introducing desired genetic modifications in several crop plants. We sought to report an improved protocol for calli production, in vitro regeneration, and a CRISPR-mediated genome editing of the phytoene desaturase (PDS) gene via a biolistic-mediated transformation system in pigeonpea. The indigenously developed construct (CcPDS_NICTK-2_pCRISPR-Cas9) harboring pigeonpea codon-optimized Cas9 and target-specific sgRNA was used for transformation in pigeonpea explants (embryonic axis and cotyledonary nodes). The addition of tailored growth regulators and silver nitrate to shoot-induction media boosted plant regeneration to about 86% (±0.04) and transformation efficiency to 46% (±0.04). Sequencing analysis revealed the incurred mutations in the native CcPDS gene, with an editing efficiency of approximately 10%. Moreover, this optimized approach can be utilized in the future to generate marker-free genome-edited plants, addressing biosafety concerns and facilitating the acceptance and commercialization of genetically improved crops.

Spalt-like transcription factor 2 (SALL2) has emerged as a potential tumor suppressor in various malignancies; however, its role in breast cancer remains underexplored. This study examines the effect of SALL2 overexpression on breast carcinogenesis, with a particular focus on the induction of apoptosis and inhibition of cell migration and invasion, using breast cancer receptor-positive and receptor-negative cell lines. Breast cancer cell lines (MCF-7 and MDA-MB-231) were transiently transfected with a SALL2 expression vector, and successful transfection was confirmed by real-time PCR and Western blot analysis. Functional assays performed included proliferation (MTT assay), wound healing, invasion (transwell Matrigel assay), apoptosis (flow cytometry), and assessment of mRNA expression levels of CDKN1A (p21), p16, PMAIP1 (NOXA), BAX, and MMP9 using quantitative real-time PCR. We demonstrated that the transient upregulation of SALL2 expression markedly inhibited cell migration and invasion, processes central to tumor metastasis. This effect was accompanied by a reduction in MMP9, a key enzyme associated with extracellular matrix degradation and metastatic potential. Furthermore, upregulated SALL2 expression significantly promoted apoptosis, as evidenced by the upregulation of pro-apoptotic genes including PMAIP1 (NOXA), BAX, CDKN1A (p21), and p16. These changes suggest that SALL2 not only impedes metastatic capacity but also enhances apoptotic signaling pathways in breast cancer cells. Importantly, the tumor-suppressive nature of SALL2 was consistent in both types of breast cancer cell lines, underscoring its broad therapeutic relevance across different breast cancer subtypes. Our findings indicate that SALL2 expression inhibits cell proliferation, migration, and invasion, while inducing apoptosis in breast cancer. These findings suggest that SALL2 may be a critical regulator of breast carcinogenesis and a potential target for therapeutics controlling tumor progression, invasion, and metastasis.

Caenorhabditis elegans is a powerful model for studying gene function and disease pathogenesis. While RNA interference effectively suppresses gene expression, CRISPR activation (CRISPRa) provides a robust tool for upregulating endogenous gene expression in C. elegans. Compared with traditional injection-based methods, feeding-based CRISPRa, similar to RNA interference, is easy, cost-effective and efficient. However, the traditional cloning workflows remain a bottleneck for high-throughput gene screening. Here, we present a pooled, one-step dual gRNA cloning protocol that enables rapid and efficient construction of gRNA expression vectors for CRISPRa. In a test case, by designing 55-mer and 54-mer primers, each containing one gRNA, we amplified and pooled 126 gRNA inserts in a single reaction pipeline. The 126 gRNA clones were completed in three pooled rounds, achieving 42%–56% coverage for each round, with remaining clones processed individually. This protocol dramatically reduces time, labor, and reagent consumption, while increasing scalability and maintaining reproducibility. It is particularly well suited for high-throughput screening of gene libraries or pathways and supports downstream applications such as phenotypic screening and lifespan analysis. This work advances CRISPRa-based functional genomics in C. elegans by providing a practical tool for large-scale gene activation studies.