CARBON Newsletter (26 March 2025) - Your Latest News About CRISPR in AgroBio

Top picks

• CRISPR-Cas9 disruption of Hookless2 (SlHLS2) in tomato revealed its central role as a key epigenetic regulator of diverse agronomic traits. Mutants showed altered architecture, infertility, pathogen susceptibility, and elevated auxin. HLS2, a member of the GCN5 family of histone acetyltransferases (HATs), modulates gene expression via histone acetylation, directly regulating SlBT1, a component of protein degradation pathways. HLS2 itself is degraded post-infection, linking chromatin dynamics to stress responses and developmental regulation.
• Using a viral-based CRISPR-Cas9 system, lettuce plants were edited to enhance nutritional value by targeting carotenoid and ascorbic acid pathways. Knockout of LCY-ε and CCD4a boosted β-carotene and zeaxanthin, while regulatory mutations in uGGP1 and uGGP2 increased ascorbic acid up to 6.9-fold. Combined edits yielded nutrient-rich plants without affecting growth, demonstrating effective multi-pathway enhancement of crop micronutrient content.

Technical advances

• Fusing Cas9 with pathogen-derived nuclear localization signals (NLSs) from Agrobacterium VirD2 and VirE2 enhanced CRISPR-Cas9 gene editing efficiency in Chlamydomonas reinhardtii. These NLSs showed strong affinity for the alga's importin alpha (IPA1), improving nuclear import and boosting editing frequency over 2.4-fold compared to SV40TAg NLS. The strategy also succeeded in Chlorella sp., demonstrating broad applicability for algal genome engineering.
• CRISPR-Cas9 editing of PlCYP81Q38 in Phryma leptostachya – a traditional Chinese medicinal herb – hairy roots disrupted sesamin biosynthesis, causing pinoresinol accumulation and loss of key lignans. Mutagenesis exceeded 79%, with frequent large-fragment deletions. Knockout also upregulated upstream lignan pathway genes. This study confirms PlCYP81Q38's role in lignan metabolism and establishes CRISPR-Cas9 as a functional genomics tool in this medicinal species.
• Using CRISPR-Cas9, researchers developed a pipeline to generate transgene-free genome-edited sorghum in the T0 generation by avoiding antibiotic selection. Targeting the PDS gene, higher editing and transgene-free rates were achieved in non-selection conditions. Up to 38.1% of edited plants lacked foreign DNA, demonstrating a viable route for rapid, transgene-free genome editing in sorghum and other vegetatively propagated crops.
• CRISPR-free TALEDs achieve mitochondrial A-to-G editing via base excision repair triggered by DddA-induced C-to-U deamination. By enhancing this pathway – using DddA6 and fusing uracil DNA glycosylase to TadA8e – researchers developed eTALED6 and eTALED6R variants with improved efficiency and specificity. These tools enable precise mtDNA edits, including pathogenic mutations, offering insights into mitochondrial gene correction.
• A compact CRISPR-Cas9 system delivered via biolistics enabled efficient multiplex genome editing in maize, including in previously non-transformable genotypes. Heritable single and stacked mutations were generated with minimal off-target effects. A novel Indel-Selective PCR assay allowed rapid genotyping of common editing outcomes. This pipeline streamlines functional genomics across diverse maize backgrounds, addressing key transformation and screening bottlenecks.
• CRISPR-Cas was used to induce reciprocal arm translocations between non-homologous chromosomes in Arabidopsis thaliana. Despite large-scale rearrangements, transgenic lines showed stable telomere length, unaltered chromatin states, and negligible transcriptional or phenotypic disruption. The findings underscore the plant's genomic resilience and support chromosome engineering as a viable tool for dissecting telomere regulation and guiding crop genome evolution.
• Engineered LbCas12a-RRV (G146R/D156R/R182V) significantly enhances CRISPR-Cas genome editing efficiency in rice and poplar, outperforming wildtype LbCas12a at both TTTV and TTV PAM sites. Its robust activity at lower temperatures addresses prior limitations, making it one of the most effective Cas12a variants for plant editing. A detailed protocol enables multiplexed vector construction for broad application across plant species.

Disease and stress control

• CRISPR-Cas9 knockout of ZmPL1 in maize enhanced drought tolerance, improving germination, survival, and antioxidant activity while reducing oxidative damage. In contrast, ZmPL1 overexpression increased drought sensitivity. Expression analysis and ABA treatments suggest ZmPL1 acts as a negative regulator of drought response, likely via modulation of ROS and stress-related pathways. Targeting ZmPL1 offers a strategy for improving drought resilience in maize.
• LbCas12a-Ultra, paired with a 7nt stem-loop reporter, increased CRISPR-Cas detection sensitivity tenfold over standard setups, enabling accurate, pre-amplification-free identification of diverse Candidatus Phytoplasma species. A multiplex strategy ensured broad pathogen coverage and a lateral flow assay supported instrument-free use. This enhanced platform offers a versatile, sensitive tool for CRISPR-Cas12a-based plant pathogen diagnostics.
• SlHP2 and SlHP3 are key cytokinin signalling genes in tomatoes. When researchers used CRISPR-Cas9 to knock them out, they observed enhanced drought tolerance under PEG-induced stress. Double mutants exhibited improved water retention, deeper root growth, and reduced stomatal density and oxidative damage. Downregulation of cytokinin-responsive and stomatal development genes under stress suggests a regulatory link. Targeting cytokinin signalling offers a strategy for improving drought resilience in crops.
• A CRISPR-dCas9-DMRcd-SunTag system was used to target methylation to nCBP-1 and nCBP-2 promoters in cassava, aiming to reduce susceptibility to cassava brown streak disease. Reduced gene expression was observed, though likely due to CRISPR interference rather than methylation. Future work will assess heritable methylation's role in resistance by segregating out editing components.
• CRISPR-Cas9 knockout of HvERF62 in barley confirmed its role in waterlogging tolerance. Mutants showed reduced chlorophyll, root activity, and aerenchyma formation under stress. HvERF62 regulates ROS balance, carbohydrate metabolism, and stress-related signalling pathways. A GWAS-identified QTL and a loss-of-function haplotype further validate its importance. The study offers genetic markers and targets for improving waterlogging resilience in barley.

Agronomic traits

• CRISPR interference using dCas9-KRAB ( (Krüppel-associated box) was applied to suppress NtbHLH47 in tobacco, revealing its role as a negative regulator of iron uptake. Targeted repression via promoter-bound gRNAs led to elevated root iron levels and metal tolerance. Upregulation of key iron-regulatory genes confirmed altered homeostasis.
• CRISPR-Cas9 knockout of HvGS3 in barley reduced plant height by over 37% and increased culm lodging resistance by 76%, linked to shorter internode cells and elevated lignin content. Suppressed gibberellin biosynthesis via GA3ox downregulation contributed to dwarfism, which was reversible by GA treatment. The study reveals HvGS3's role in stem structure and hormone regulation, offering the potential for lodging-resistant barley breeding.
• CRISPR-Cas9 was used to insert a 35S promoter upstream of OsNRAMP7 in the elite rice cultivar TBR225, driving its overexpression. Edited lines showed up to 72% higher grain iron without altering zinc, cadmium levels, or key agronomic traits. The study supports OsNRAMP7 overexpression as a targeted, agronomically safe strategy for iron biofortification in rice.
• CRISPR-Cas9 HDR was used to introduce point mutations in the native CcEPSPS gene of pigeon peas, reducing glyphosate binding affinity while preserving enzyme function. Edited T2 plants showed stable inheritance, high glyphosate tolerance, and unaltered physiological and agronomic performance post-treatment. This represents the first precise gene-editing in pigeon peas, enabling in-crop glyphosate use for effective, sustainable weed management.

Industry

• Cibus, a leader in gene-edited productivity traits, has demonstrated enhanced Sclerotinia resistance in canola using its CRISPR-Cas-assisted RTDS platform. The trait is part of a broader pipeline with four distinct gene-edited resistance strategies, two of which are already in field trials. The results support the commercial viability of durable fungal resistance through precise, timebound gene editing.
• Cibus welcomes the proposed EU rules exempting 'conventional-like' gene-edited crops from GMO regulation. The company's CRISPR-Cas-assisted RTDS traits in rapeseed – enhancing seed retention, disease resistance and nutrient efficiency – align with the proposed legislation that identifies a 'conventional-like' category of NGT-1 products that would be regulated as conventional varieties.
• Cibus has announced full-year 2024 financial results with a net loss of $283 million and $14 million in cash. The large loss is mainly due to a goodwill impairment, and despite the low cash balance, the company expects to have sufficient funding until the second half of 2025.

Detection

• A CRISPR-Cas12a assay, paired with recombinase polymerase amplification, enables rapid, on-site detection of the fungal pathogen Diaporthe helianthi in sunflower within 45 minutes at 37°C. Achieving sensitivities down to 0.1 pg/μL, it outperforms real-time PCR by ~100-fold. Compatible with lateral flow and fluorescence readouts, the system supports field diagnostics for early disease management and quarantine surveillance.

Reviews

• Enhancing Sugar Crop Resilience to Abiotic Stress Using CRISPR/Cas Tools. This review explores the application of CRISPR-based genome editing technologies to enhance abiotic stress tolerance in sugar crops, with emphasis on regulatory networks, key transcription factors, transgene-free strategies, and prospects for sustainable crop improvement.
• CRISPR–Cas applications in agriculture and plant research. This review outlines the expanding CRISPR-Cas toolkit for crop improvement, highlighting emerging Cas variants, base and prime editing, and delivery strategies. It details applications in genome, epigenome, and transcriptome engineering, including promoter editing, chromosome engineering, and de novo domestication.
• Novel application of ribonucleoprotein-mediated CRISPR-Cas9 gene editing in plant pathogenic oomycete species. This review explores the development and application of CRISPR-Cas9 gene editing in forest pathogenic Phytophthora species, highlighting the successful generation of gene-edited P. cactorum mutants with reduced fungicide resistance and the challenges encountered with P. ramorum.
• Molecular breeding of pigs in the genome editing era. This review summarises advances in porcine molecular breeding, focusing on CRISPR-based strategies to identify resistance loci and improve production traits. It highlights the limitations of traditional breeding and the potential of genome editing to overcome species barriers and accelerate genetic gains.
• Synthetic gene circuits in plants: recent advances and challenges. This review explores the design, application, and future prospects of synthetic gene circuits in plants, highlighting recent advances, current challenges, and opportunities for improving programmable gene expression through quantitative analysis, high-throughput testing, and modelling.
• CRISPR/Cas9: a sustainable technology to enhance climate resilience in major Staple Crops. This review explores the application of CRISPR-Cas9 genome editing to develop climate-resilient cereal crops, highlighting key target traits and their potential to enhance wheat, rice, and maize tolerance to environmental stress.