The core idea behind multiplex genome editing is deceptively simple. Where early genome editing tools such as zinc finger nucleases and transcription activator-like effector nucleases, known as ZFNs and TALENs, required a new engineered protein for every DNA target, CRISPR-based systems use small guide RNAs that can be reprogrammed cheaply and quickly. Multiplexing takes this a step further by deploying two or more guide RNAs simultaneously, allowing researchers to edit multiple loci in a single experiment, sometimes down to the single-nucleotide level. Because of this superior precision and feasibility, the authors note, CRISPR-MGE has largely displaced TALENs and ZFNs in plant laboratories worldwide, becoming the default platform for both functional genomics and applied crop improvement.

Delivering many guide RNAs at once posed an early engineering challenge, and the review catalogs the creative solutions that emerged. The most straightforward approach uses individual expression cassettes, with each guide RNA driven by its own promoter, an effective but bulky strategy that becomes unwieldy as target numbers grow. More elegant systems exploit the cell’s own machinery. The endogenous tRNA-processing system, for example, allows researchers to string multiple guide sequences together in a single synthetic gene, flanked by tRNA sequences that the plant itself cleaves apart to release mature guides. This strategy has been demonstrated in maize, rice, and cabbage, among other species. Alternative approaches borrow from bacterial immunity and RNA biology: the CRISPR-associated endoribonuclease Csy4 can process a polycistronic transcript into individual guide RNAs, while self-cleaving ribozymes, first characterized as simple RNA enzymes in the late 1980s, can be placed around guide sequences to achieve the same result.

Beyond these foundational strategies, the review highlights newer advances that have refined multiplex editing into what the authors describe as a powerful, efficient, and robust toolkit. The Cas12a enzyme, formerly known as Cpf1, has proven especially valuable because it processes its own CRISPR RNA arrays, naturally lending itself to multiplexing while recognizing a different protospacer-adjacent motif than Cas9 and thereby expanding the range of editable genomic sites. Engineered Cas12a variants with relaxed PAM requirements and temperature tolerance have extended editing into rice, maize, and tomato under conditions where earlier systems faltered. At the same time, ultra-multiplexing platforms now permit the simultaneous targeting of dozens of loci, and orthogonal systems such as CRISPR-Combo allow genome editing and transcriptional activation to proceed in parallel within the same cell, a capability demonstrated in tomato and other species.

Perhaps the most consequential shift described in the review is the move toward transgene-free editing. Conventional CRISPR experiments introduce DNA constructs that integrate into the plant genome, leaving behind foreign sequences that complicate regulation and public acceptance. DNA-free alternatives circumvent this problem entirely. Preassembled Cas9 ribonucleoproteins, complexes of purified protein and guide RNA, can be delivered into plant cells by PEG-mediated transfection of protoplasts, lipofection, particle bombardment, or cationic lipid nanoparticles, producing edits that are inherited while the editing machinery itself is rapidly degraded and never integrated. The review cites successful DNA-free editing in potato, maize, canola, soybean, citrus, carrot, and tomato, including the generation of transgene-free canker-resistant sweet orange using Cas12a ribonucleoproteins. Virus-based guide RNA delivery systems offer another route, with vectors derived from potato virus X and other plant viruses shuttling guide RNAs through the plant to generate heritable edits without stable transgene integration.

Why does multiplexing matter so much for stress biology? The answer lies in the architecture of plant stress responses. Transcription factors, the DNA-binding proteins that switch suites of target genes on or off, sit at the hubs of regulatory networks that coordinate a plant’s reaction to abiotic stresses such as drought, salinity, cold, and heat, as well as biotic attacks by pathogens and pests. These networks are notoriously redundant and interconnected: single-gene knockouts often produce subtle or no phenotypes because paralogs and parallel pathways compensate. Multiplex editing cuts through this redundancy by disabling entire gene families or combinations of regulators at once, revealing the true structure of the network. The review emphasizes that this capacity makes CRISPR-MGE ideal for elucidating the function of transcription factors, the key molecular players regulating diverse plant responses, especially within stress pathways.

The empirical record assembled in the review illustrates the point across crops and stress types. In rice, knockout of the OsbHLH024 transcription factor improved salt stress resistance, while editing of NAC-family members such as OsNAC15 and OsNAC45 has illuminated their roles in drought, salt, and abscisic acid responses. In tomato, CRISPR-Cas9 mutagenesis of SlNPR1 reduced drought tolerance, confirming its positive regulatory role, while disruption of SlCBF1 diminished chilling tolerance and loss of SlMYC2 compromised methyl jasmonate-induced fruit resistance to the gray mold pathogen Botrytis cinerea. In wheat, simultaneous editing of the three homoeoalleles of TaEDR1 enhanced powdery mildew resistance, a landmark demonstration of why polyploid crops demand multiplex approaches, since useful traits often require hitting all redundant copies at once. Multiplex editing of BnWRKY11 and BnWRKY70 in oilseed rape, and of stress-linked regulators in poplar and grapevine, round out a picture of a technology operating across the plant kingdom.

The toolkit’s reach extends beyond simple knockouts. Multiplex platforms now support base editing, which converts individual DNA letters without cutting both strands, and prime editing, which can install precise sequence changes; both have been deployed in multiplex form in rice, wheat, and maize for agronomically important genes. Nuclease-dead Cas9 fused to activation or repression domains, such as the VP64 activator or the SRAX repressor domain, enables transcriptional regulation of target genes without altering their sequence, and epigenome editing fusions, such as a histone acetyltransferase tethered by dCas9, have improved drought tolerance in Arabidopsis. Metabolic engineering applications, from boosting gamma-aminobutyric acid and lycopene in tomato to raising carotenoid and isoflavone levels in rice and soybean, demonstrate that multiplex editing can reconfigure entire biosynthetic pathways, the same logic needed to tune stress-responsive hormone and antioxidant networks.

The review also confronts the practical bottlenecks that still separate laboratory success from farmers’ fields. Plant regeneration remains a limiting step for many species and genotypes, though morphogenic regulators such as GRF-GIF chimeric proteins and growth-regulating factors are boosting transformation efficiency in crops like sorghum and wheat. Quantifying editing outcomes has grown more rigorous with droplet digital PCR and microfluidic chip-based digital PCR, which allow precise measurement of edit frequencies, an important safeguard given that off-target mutations and variable on-target activity remain persistent concerns. Regulatory landscapes, which differ between process-based and product-based frameworks across jurisdictions, will shape how quickly edited stress-tolerant varieties reach the market, and the authors implicitly position transgene-free methods as a way to ease that transition.

Taken together, the review delivers a clear message to the plant science community: advanced multiplex genome editing is no longer an experimental luxury but the central instrument for decoding and redesigning stress-responsive transcription factor networks. As the authors argue, deploying these tools for the functional characterization of stress-responsive transcription factors holds genuine potential to accelerate crop improvement at a moment when rising salinity, erratic rainfall, and emerging pathogens threaten harvests across South Asia and beyond. The convergence of ultra-multiplexing, orthogonal regulation, DNA-free delivery, and precision base and prime editing means that researchers can now ask, and answer, questions about genetic redundancy and network logic that were unanswerable only a few years ago. The next generation of climate-resilient crops, the review suggests, will be written not one gene at a time, but in whole regulatory paragraphs.

Subject of Research: CRISPR multiplex genome editing of stress-responsive transcription factor networks for crop improvement

Article Title: Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement

Article References: Sharma, N. N., Kumari, A., Vashisht, I., & Sharma, M. K. (2026). Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement. Plant Cell Reports, 45(9), Article 277. https://doi.org/10.1007/s00299-026-03956-w

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03956-w

Keywords: CRISPR, multiplex genome editing, transcription factors, stress response, crop improvement, Cas12a, base editing, prime editing, transgene-free editing, drought tolerance, salt tolerance, plant biotechnology