MicroRNAs, or miRNAs, are single-stranded RNA molecules typically 21 to 24 nucleotides in length. Although they do not encode proteins, they perform a crucial regulatory function by binding to messenger RNA targets and either cleaving them or blocking their translation. In doing so, they fine-tune the expression of transcription factors, hormone signaling components and transporter genes that govern virtually every aspect of plant life, from root architecture and flowering time to drought tolerance and grain filling. A single miRNA can regulate multiple genes within the same pathway, which makes these molecules extraordinarily powerful levers for crop improvement. Environmental conditions can reshape miRNA expression profiles, and in turn the plant’s stress responses, offering a dynamic regulatory layer that breeders have only begun to exploit.

The review, led by Kasanaboina Krishna of the International Crops Research Institute for the Semi-Arid Tropics and colleagues, synthesizes evidence from across the major cereals and emerging millet studies to build a millet-focused regulatory framework. The authors distinguish between conserved grass-wide miRNA modules and millet-specific candidates, and they are candid about the state of the field: most millet miRNA research to date has been limited to computational prediction and expression profiling, while rigorous functional validation remains scarce. Degradome sequencing evidence, tissue-specific regulatory maps and field-relevant genotype-by-environment analyses are still largely missing, meaning that many of the most exciting candidates remain hypotheses rather than established tools.

Nevertheless, the evidence that does exist is compelling. In foxtail millet, miR394 has been shown to positively regulate drought resistance, with upregulation after treatment with methyl jasmonate, ethephon, salicylic acid and abscisic acid, and improved germination rates and root lengths in response. A member of the miR396 family, SimiR396d, targets the growth-regulating factor gene SiGRF1, and its overexpression enhances both root growth and drought tolerance, directly linking miRNA-mediated developmental regulation to stress adaptation. In pearl millet, one of the most drought-tolerant cereals known, researchers identified 61 novel miRNAs under high vapor pressure deficit, with families including miR167, miR172, miR396 and miR399 implicated in root physiology and abiotic stress responses. Sorghum studies have revealed 80 individual miRNAs responding to drought, heat and combined stress, including eight novel stress-responsive families.

Salinity tolerance offers another striking example. In finger millet, the Eco-miR169–EcNF-YA13 regulatory module has been identified as a key determinant of dehydration and salinity tolerance; the transcription factor EcNF-YA13 supports stress tolerance but is suppressed by Eco-miR169, suggesting that modulating this miRNA could relieve the repression and enhance tolerance. Earlier work in the same species identified 48 conserved and 35 novel salinity-responsive miRNAs, with several families upregulated more than tenfold in tolerant genotypes. In pearl millet, small RNA sequencing revealed 95 salinity-responsive miRNAs targeting 448 genes, many involved in auxin responses, hinting that miRNA-mediated hormone regulation underpins the crop’s remarkable salt resilience.

Beyond stress tolerance, miRNAs appear central to the nutritional traits that make millets so valuable as nutri-cereals. Finger millet is prized for its calcium content, pearl millet for iron and zinc, kodo millet for dietary fiber and phenolics, and fonio for sulfur-containing amino acids. Yet the direct links between specific miRNAs and grain micronutrient accumulation remain under-investigated. One notable exception comes from pearl millet, where pgl-miR159 was identified as a candidate associated with iron metabolism during a broader search for grain iron and zinc genes. In rice, high-iron transgenic lines showed downregulation of root-specific miRNAs that in turn upregulated key transporters such as OsYSL15, OsFRO2 and OsIRT1, boosting iron and zinc uptake. The review argues that identifying orthologous transporter–miRNA modules in millets could provide a mechanistic framework for biofortification, provided the interactions are experimentally validated.

The translational toolkit for moving from candidate miRNAs to improved cultivars is now substantial. The authors propose a roadmap combining tissue- and stage-resolved miRNA atlases with target validation through degradome sequencing, RNA Ligase-Mediated Rapid Amplification of cDNA Ends and reporter assays. Functional intervention platforms include short tandem target mimics, which sequester endogenous miRNAs to relieve repression of beneficial target genes; artificial miRNAs, which deliver highly specific gene knockdowns with minimal off-target effects; and CRISPR/Cas-mediated editing of miRNA loci, promoters or target recognition sites. Proof-of-concept studies in other cereals show the power of these approaches: editing the miR396 binding sites in rice OsGRF4 and OsGRF8 derepressed growth and boosted grain size, while CRISPR-induced mutations in the miR156 recognition element of wheat TaSPL13 improved grain number, size and architecture.

Integration with breeding pipelines is the critical next step. miRNA-derived molecular markers, first developed in Brassica and rice, capture regulatory variation rather than merely neutral structural differences, linking markers directly to traits such as stress tolerance and yield stability. In foxtail millet, researchers designed 66 primer pairs from conserved pre-miRNA sequences with high cross-genera transferability, underscoring their promise as functional genotyping tools. The review also highlights the potential of machine learning and digital miRNA twins, computational models trained on sequence features and expression data to predict stress-responsive miRNAs and simulate how edited or introgressed miRNA modules would perform across drought cycles, heatwaves and nutrient-poor soils before any field trial. Embedding miRNA target interactions into crop simulation platforms could dramatically shorten breeding cycles for climate-resilient varieties.

The regulatory and ecological dimensions are not ignored. India’s 2022 guidelines exempt site-directed nuclease 1 and 2 genome-edited plants from GMO-style environmental risk assessment once vector sequences are segregated, opening a practical pathway for non-transgenic miRNA edits. At the same time, the authors note that plant miRNAs can move within and between organisms, a consideration for environmental safety even in cisgenic edits. Benchmarks for nutritional outcomes already exist: Indian biofortified pearl millet targets of at least 42 milligrams of iron and 32 milligrams of zinc per kilogram of grain, achieved without sacrificing yield. Rapid screening systems built around the model grass Setaria viridis, including spike-dip transformation, protoplast assays and foxtail mosaic virus vectors for virus-induced gene silencing, could accelerate functional validation before the best constructs move into stable millet genotypes for multi-environment testing.

The broader message is one of urgency and opportunity. Millets already possess C4 photosynthesis, deep root systems, compact stature and strong antioxidant defenses that allow them to thrive where major cereals fail. Their inherent micronutrient richness makes them ideal testbeds for miRNA-guided climate-smart breeding. What is missing is the systematic functional validation that would transform descriptive miRNA catalogues into experimentally confirmed regulatory networks. If the roadmap laid out in this review is followed, building comprehensive miRNA atlases, mining landrace and wild-relative diversity for novel miRNA alleles, deploying CRISPR and target mimicry tools, and integrating validated miRNA-trait associations into genomic selection models, the humble millet could become a global model for genetic resilience, nutritional density and smart agriculture, delivering climate-proof, micronutrient-dense grain to the farmers and consumers who need it most.

Subject of Research: MicroRNA regulatory networks controlling climate resilience and nutritional traits in millet crops

Article Title: Harnessing microRNA regulatory networks for engineering climate resilience and nutritional enhancement in millets

Article References: Krishna, K., Habyarimana, E., Jamedar, H. R., VG, I. L., Chavan, S., Prasad, B. V. V., Mohan, Y. C., Edukondalu, B., & Ceasar, S. A. (2026). Harnessing microRNA regulatory networks for engineering climate resilience and nutritional enhancement in millets. Stress Biology, 6(1), Article 57. https://doi.org/10.1007/s44154-026-00332-2

Image Credits: AI Generated

DOI: 10.1007/s44154-026-00332-2

Keywords: microRNAs, millets, climate resilience, drought tolerance, salinity stress, biofortification, CRISPR genome editing, foxtail millet, pearl millet, finger millet, nutritional quality, molecular breeding

CRISPR systems do not generally cut DNA at arbitrary locations. Their molecular targeting depends on two interacting components: a guide sequence that pairs with the chosen DNA site and a nearby short motif known as a protospacer adjacent motif, or PAM. The PAM acts as a molecular permission signal, allowing a CRISPR nuclease to bind and activate only when the correct neighboring sequence is present. Cas12a, a nuclease widely used for genome editing and nucleic-acid detection, has distinctive advantages, including its ability to recognize a PAM positioned differently from the one used by the more familiar Cas9 enzyme. But those advantages come with a limitation: commonly used Cas12a proteins generally require relatively long or restrictive PAM sequences. A desired genetic target may therefore be perfectly suited to the guide RNA yet remain inaccessible because the adjacent DNA does not satisfy the enzyme’s PAM rule.

Prime editing adds another layer of precision to this problem. Rather than relying on a conventional double-strand break followed by the cell’s repair machinery, prime editing typically uses a modified Cas protein fused to a reverse transcriptase. The Cas component nicks one DNA strand, while a specialized prime-editing guide RNA both directs the complex to the target and carries a template encoding the intended change. The reverse transcriptase copies that template into the DNA, creating a modified strand that the cell can incorporate into the genome. In principle, this arrangement allows researchers to install single-base substitutions, short insertions and deletions while avoiding the potentially disruptive double-strand breaks associated with standard CRISPR cutting. Yet prime editing is only as versatile as the nuclease that delivers it: if the Cas enzyme cannot recognize a nearby PAM, the edit may be impossible or require a less favorable target site.

To loosen that constraint, the team examined and engineered variants related to four Cas12a enzymes: LbCas12a, AsCas12a, MbCas12a and FnCas12a. Their goal was to create PAM-flexible proteins that remained active inside mammalian cells rather than merely showing altered biochemical behavior in a test tube. Among the variants tested, flexiAsCas12a—derived from AsCas12a—proved the most effective in the reported experiments. The researchers found that the variant could extend Cas12a’s recognized PAM repertoire to include sequences described as NATN, NCCN and GTCN. In this notation, N represents any nucleotide, while the specified letters impose only partial constraints on the four-base motif. Compared with a highly restrictive PAM requirement, these patterns make more sites across the genome potentially addressable by the nuclease.

The significance of the expanded recognition rules is not simply that three additional sequence patterns have been added to a catalog. PAM availability is distributed unevenly across DNA, and the position of a PAM relative to a disease-associated mutation, regulatory element or coding sequence can determine whether an edit is practical. A target may require the guide and editing machinery to approach from a particular direction, or the desired alteration may fall within a limited distance from the nick introduced by the nuclease. More permissive PAM recognition increases the odds that a usable site exists in the right genomic context. It could also reduce the need to redesign an experiment around a nearby, imperfectly positioned target. However, broader recognition must be balanced against the risk of unintended activity: an enzyme that accepts more PAMs may encounter more possible sites, making rigorous off-target testing essential before therapeutic applications can be considered.

The study also connects PAM flexibility to a less conventional form of prime editing. The researchers used currently available Cas12a variants with relaxed PAM recognition—impLbCas12a, flexiAsCas12a and enAsCas12a—to develop circular RNA-guided split prime editors. In a split editor, the molecular machinery is divided into separate components rather than delivered as one large protein or complex. This strategy can help address the size limitations that complicate the delivery of genome-editing systems, particularly with viral vectors, whose cargo capacity is restricted. Circular RNAs are RNA molecules whose ends are joined, a structure that can offer greater resistance to degradation than conventional linear RNA. By combining circular RNA guidance with split prime-editing architecture, the researchers sought to create systems that could function at targets carrying non-canonical PAMs—sequences that would not ordinarily be accepted by standard Cas12a tools.

The experiments validated the functionality of these circular RNA-guided split editors on non-canonical PAM sequences, according to the study. That result is important because it demonstrates more than a theoretical change in target recognition: the engineered enzymes could be incorporated into a working prime-editing system in mammalian cells. The work does not, however, establish that every newly recognized target will be edited with equal efficiency, nor does it show that the approach is ready for clinical use. Genome editing performance depends on many variables, including chromatin structure, guide-RNA design, the exact DNA change being attempted, cellular repair pathways and the concentration and duration of the editing components. The study’s central achievement is therefore an expansion of the addressable target space, rather than a universal solution to the technical and safety challenges facing prime editing.

Cas12a’s biology may make these variants attractive for applications beyond prime editing as well. Cas12a enzymes are already used in nucleic-acid detection because their activity can produce detectable signals after target recognition. In genome manipulation, their guide-RNA processing properties and targeting geometry differ from those of Cas9, providing alternative ways to design multiplexed or directionally constrained editing experiments. A broader PAM range could allow researchers to choose among more Cas12a configurations according to the genomic site and desired outcome. The new variants may also be useful when a Cas9-based tool has unfavorable off-target behavior or cannot reach a sequence in the required orientation. Whether flexiAsCas12a or related enzymes outperform established systems will depend on head-to-head measurements of editing yield, product purity, unintended edits, cellular toxicity and delivery efficiency.

The authors describe flexiAsCas12a as a new addition to the expanding collection of Cas12a PAM variants, with the broader objective of making more genomic sequences accessible to precision editing. The work was conducted by researchers affiliated with the HUN-REN Research Centre for Natural Sciences, the University of Szeged, the Biological Research Centre, Eötvös Loránd University, Semmelweis University and Hungarian biotechnology organizations. The article was made available as an early peer-reviewed, accepted version carrying a permanent DOI, with the publisher noting that it may later be replaced by a final version of record. As the field moves toward increasingly programmable genome modification, the practical importance of the advance will be determined by how reliably these flexible nucleases edit difficult targets while preserving the selectivity that makes prime editing appealing. For now, the study offers a molecular workaround to one of CRISPR’s most persistent limitations: the short sequence beside a target that can decide whether the target is editable at all.

Pennycress is inherently a hardy and fast-growing plant renowned for its freeze tolerance and rapid life cycle, characteristics that make it an ideal candidate for off-season cultivation. Yet, despite these advantages, wild forms of pennycress exhibit traits that traditionally hinder their agricultural viability, such as high seed dormancy and seed coat features contributing to seedling emergence problems and potential weediness. The research team overcame these barriers by deploying highly targeted CRISPR-mediated mutations to tweak and combine key domestication traits while ensuring minimal negative impact on seed production.

One of the most striking outcomes of this genetic refinement is the dramatic reduction of seed glucosinolate levels, compounds that are naturally abundant in many brassicas but can negatively impact the nutritional quality and safety of seeds for food and feed applications. By inactivating genes encoding R2R3-MYB transcription factors, specifically MYB28 (also designated as HAG1), alongside mutations in the basic helix–loop–helix transcription factor MYC3, the engineered pennycress varieties showed a remarkable 75% decrease in seed glucosinolate content. This “double-low” profile mirrors the advantageous traits found in canola, where low erucic acid and reduced glucosinolates have already revolutionized oilseed utility.

In parallel, the researchers tackled the issue of seed coat characteristics and seed dormancy through knocking out the basic helix–loop–helix transcription factor TRANSPARENT TESTA8 (TT8). This mutation significantly attenuated seed dormancy and weakened seed coat defenses, effectively reducing the weediness potential of pennycress by curbing unwanted re-emergence of volunteer plants in subsequent crop cycles. This breakthrough ensures that pennycress not only fits seamlessly as an intermediate crop but also poses minimal risk of becoming an invasive threat in managed agricultural landscapes.

The cumulative effect of stacking these targeted mutations has produced high-yielding, low-carbon-intensity pennycress varieties tailored for the unique temporal niche between two full-season summer crops such as corn and soybean. Integrating pennycress into existing cropping systems enables farmers to harvest three cash crops within a two-year span, effectively transforming the previously idle winter or early spring fallows into productive land with tangible economic benefits. Beyond yield improvements, this approach also confers substantial ecosystem services akin to traditional cover crops, including soil erosion reduction, increased carbon sequestration, and enhanced biodiversity.

This bioengineered pennycress heralds a new era of crop diversification essential for global food security and climate change mitigation. By converting what was once considered marginal or underutilized land into productive farmland with reduced environmental footprint, the innovation aligns perfectly with the urgent demands of sustainable intensification in agriculture. It provides a versatile platform for producing renewable biofuels and plant-based oils essential for food and industrial applications, thereby enhancing resource efficiency.

The utilization of CRISPR–Cas9 technology in this context exemplifies the power of precision breeding enabled by modern molecular genetics. Unlike traditional breeding, which can be laborious and time-consuming, genome editing allows direct modulation of specific genes responsible for domestication traits without introducing extraneous genetic material. This precise approach facilitated rapid iteration and stacking of multiple favorable traits to synergistically improve crop performance.

Moreover, the domesticated pennycress varieties possess seed fiber compositions optimized for human and animal consumption, achieved by selecting mutations that lower seed fiber content while maintaining seed integrity. This balance is critical for ensuring that the seeds can be processed efficiently into oils, meals, and other value-added products within existing agricultural supply chains without requiring mechanical or biochemical modifications.

The success in domestication also underscores the importance of transcription factors as master regulators for complex traits such as seed chemistry and dormancy. Modulating MYB and bHLH family transcription factors demonstrates that subtle shifts in gene expression networks can have profound phenotypic consequences, allowing for the fine-tuning of multiple interrelated traits simultaneously. This insight can inspire similar approaches in other orphan or underutilized crops with potential untapped benefits.

By introducing agronomically valuable traits without compromising the ecological resilience and rapid growth characteristics of pennycress, the study provides a model for sustainable crop development. The integration of this engineered pennycress into crop rotations potentially reduces reliance on synthetic inputs and mitigates greenhouse gas emissions associated with mono-cropping high-intensity crops. This approach also alleviates pressure on land-use expansion, thereby protecting natural ecosystems.

The research not only provides immediate solutions but opens avenues for further improvements, such as refining seed oil compositions tailored to industrial uses or biofuel specifications, enhancing stress tolerance to diverse climates, and optimizing plant architecture for mechanized harvesting. Given pennycress’s short generation time, continuous genetic advancements can be rapidly incorporated to fine-tune performance across different geographies and cropping systems.

This breakthrough also reflects broader trends in plant science where de novo domestication is gaining traction as a viable strategy to accelerate crop diversification. Utilizing genome editing to transform wild species into cultivated crops suited for modern agricultural needs expands the toolbox for breeders grappling with challenges posed by climate change, population growth, and food system sustainability.

In conclusion, the creation of domesticated, genome-edited pennycress strains represents a landmark achievement in agricultural biotechnology with profound implications. By providing a high-yield, low-input, and environmentally compatible intermediate crop, the innovation effectively converts dormant farmland into a productive asset that supports both economic and ecological objectives. This progress exemplifies the promise of synthetic biology to design the next generation of crops tailored for a sustainable future.

Subject of Research: Development and de novo domestication of the oilseed crop pennycress using CRISPR–Cas9 to introduce beneficial agronomic traits.

Article Title: Creating a new oilseed crop, pennycress, by combining key domestication traits using CRISPR genome editing.

Article References:
Gautam, B., Jarvis, B.A., Esfahanian, M. et al. Creating a new oilseed crop, pennycress, by combining key domestication traits using CRISPR genome editing. Nat. Plants (2026). https://doi.org/10.1038/s41477-025-02202-7

Image Credits: AI Generated

DOI: https://doi.org/10.1038/s41477-025-02202-7

The oncogene Myc plays a pivotal role in regulating cell proliferation, metabolism, and apoptosis, but its dysregulation unleashes a torrent of aberrant cellular processes culminating in malignancy. Despite extensive research efforts, effective therapeutic strategies to counter Myc-driven cancers remain elusive because Myc itself is considered “undruggable.” Therefore, functional genetic screens aimed at uncovering synthetic lethal partners or tumor suppressors that cooperate with Myc represent a strategic pathway toward translational breakthroughs.

Exploiting the revolutionary CRISPR-Cas9 genome editing technology, Potts and colleagues adopted an innovative in vivo screening approach that surpasses the limitations of traditional in vitro models. By introducing a genome-wide CRISPR library directly into living lymphoma models, the research team interrogated the entire murine genome for genes whose loss potentiates or suppresses Myc-driven tumorigenesis. This exhaustive, unbiased strategy empowers the discovery of physiologically relevant tumor suppressors acting within the intact cellular and microenvironmental context of lymphoma development.

The GATOR1 complex, comprising DEPDC5, NPRL2, and NPRL3, emerged as a top hit from these screens, pinpointing it as a critical tumor suppressor nexus. Prior to this study, GATOR1 was chiefly recognized for its canonical role in nutrient-sensing and mTORC1 signaling—a pathway often hijacked by cancer cells to sustain unchecked growth. The discovery that GATOR1 loss accelerates Myc-driven lymphoma progression substantiates a model where GATOR1 functions as a cellular brake to metabolic reprogramming induced by Myc oncogene activation.

Intriguingly, mechanistic investigations revealed that disruption of GATOR1 components unleashes hyperactive mTORC1 signaling, culminating in elevated anabolic metabolism and augmented tumor cell proliferation. This hyperactivation compromises cellular homeostasis and favors a metabolic environment conducive to oncogenesis. These findings underscore the functional interplay between metabolic pathways and oncogenic transcription factors, highlighting the therapeutic potential of targeting mTORC1 downstream effects in Myc-driven malignancies.

Using sophisticated genetic mouse models and RNA sequencing, the study delineated how GATOR1 loss reshapes the transcriptional landscape of lymphoma cells. Specifically, GATOR1 deficiency amplifies expression of genes involved in ribosome biogenesis, nucleotide synthesis, and mitochondrial function—hallmarks of a hyperproliferative state. This transcriptional reprogramming converges on amplifying Myc’s oncogenic output, thus establishing a feed-forward loop that fosters lymphoma aggressiveness.

The translational implications are profound. mTORC1 inhibitors, such as rapamycin analogs, are already clinically available, and this study provides a strong rationale for their repurposing in subsets of lymphoma patients whose tumors exhibit compromised GATOR1 function. Moreover, these findings advocate for the development of precision medicine strategies that integrate tumor genetic profiling with metabolic vulnerabilities.

Importantly, the in vivo CRISPR screening methodology demonstrated here sets a new standard for cancer functional genomics. By preserving the tumor microenvironment and immune interactions, this platform yields findings with greater clinical relevance than conventional cell culture-based screens, which often fail to recapitulate the complexity of tumor biology in living organisms.

These insights into GATOR1’s tumor-suppressive role also prompt reevaluation of metabolic checkpoints in oncogenesis more broadly. Given that Myc deregulation occurs across a wide spectrum of cancers, it is plausible that GATOR1-mediated mTORC1 control represents a conserved tumor suppressive mechanism beyond lymphoma, warranting broader investigation.

The work also raises intriguing questions about how metabolic stress and nutrient sensing intersect with oncogenic signaling pathways. The GATOR1 complex, by virtue of its nutrient-sensing capabilities, may link extracellular environmental cues with intracellular oncogenic circuits, thereby influencing cancer cell adaptability and survival during tumor progression.

Moreover, this study exemplifies the power of systems biology approaches that integrate genetic screening, metabolic analysis, and transcriptional profiling to decode cancer vulnerabilities. Such holistic frameworks are essential to unravel the multifaceted nature of oncogene addiction and resistance mechanisms that underlie clinical challenges.

While the therapeutic landscape for Myc-driven lymphoma remains challenging, the identification of GATOR1 as a tumor suppressor provides a concrete molecular handle for drug development efforts. It is conceivable that combinatorial regimens targeting both Myc-associated transcriptional programs and mTORC1 signaling could yield synergistic anti-tumor effects, potentially overcoming resistance that plagues monotherapies.

This research also contributes to our understanding of how cancer cells exploit metabolic rewiring to thrive under oncogenic stress. By targeting the metabolic dependencies forged by Myc overactivation, future interventions may achieve higher specificity and reduced toxicity.

Beyond cancer, the role of the GATOR1 complex in nutrient sensing and metabolism suggests broader physiological implications, raising the possibility that its dysfunction could contribute to other pathological states linked to mTOR dysregulation. This opens a fertile area for further biomedical inquiry.

As genome editing tools continue to evolve, the integration of in vivo CRISPR screens with single-cell sequencing and spatial transcriptomics promises to accelerate discovery of tumor suppressors with unprecedented resolution. Studies like this herald a new era where functional genomics merges seamlessly with cancer therapeutics.

In summary, Potts, Mizutani, Deng, and colleagues have delivered a seminal contribution by revealing GATOR1 as a pivotal tumor suppressor within Myc-driven lymphoma, strategically connecting metabolic regulation with oncogenic transcription. Their work not only charts new territory in cancer biology but also lays the foundation for novel therapeutic strategies that may someday translate into tangible benefits for patients afflicted by these aggressive malignancies.

Subject of Research: Identification of tumor suppressor genes in Myc-driven lymphoma using genome-wide in vivo CRISPR screens

Article Title: Genome-wide in vivo CRISPR screens identify GATOR1 complex as a tumor suppressor in Myc-driven lymphoma

Article References:
Potts, M.A., Mizutani, S., Deng, Y. et al. Genome-wide in vivo CRISPR screens identify GATOR1 complex as a tumor suppressor in Myc-driven lymphoma. Nat Commun 16, 7582 (2025). https://doi.org/10.1038/s41467-025-62615-y

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