The review’s abstract describes recent progress in identifying novel Cas orthologs and engineering Cas9 variants to improve editing fidelity, expand target ranges and reduce off-target effects. It also reports that structural and functional optimization of single-guide RNAs has improved target-binding affinity, stability and formation of the Cas9-sgRNA complex. High-fidelity Cas9 derivatives, base editors and prime editors are presented as enabling precise single-nucleotide substitutions and small insertions or deletions without producing double-stranded DNA breaks.

Delivery remains a central focus. According to the abstract, viral vectors, lipid nanoparticles and ribonucleoprotein electroporation have advanced CRISPR-mediated editing across in vitro, ex vivo and in vivo models. The review highlights therapeutic milestones reported for sickle cell disease, β-thalassemia and cystic fibrosis, where the abstract states that long-term clinical benefits have been documented. It also identifies a separate research role for CRISPR-Cas9 in diabetes: modeling disease mechanisms, identifying molecular pathways involved in glucose homeostasis and supporting development of cellular and gene-based therapies.

This is a review rather than a new clinical trial, laboratory study or protocol, and the available evidence consists of the article’s abstract and publisher information rather than the full text. The abstract does not provide study populations, sample sizes, quantitative editing efficiencies, delivery-performance measures, follow-up periods, safety outcomes or a systematic assessment of the cited clinical evidence. Consequently, the reported therapeutic milestones and broader translational claims are summarized here as the review’s conclusions; the available source does not support independent verification of their magnitude or applicability to individual patients.

Subject of Research: CRISPR-Cas9 genome editing technologies, delivery systems and therapeutic applications

Article Title: CRISPR–Cas9 genome editing: technological advances, delivery strategies and precision engineering of primary cells for therapeutic application in genetic disorders and diabetes mellitus

Article References: Islam, M. N., Islam, M. M., Feng, H., Bellah, S. F., Zaman Miah, M. M., & Hossain, M. S. (2026). CRISPR–Cas9 genome editing: technological advances, delivery strategies and precision engineering of primary cells for therapeutic application in genetic disorders and diabetes mellitus. Molecular Biology Reports, 53(1), Article 1589. https://doi.org/10.1007/s11033-026-12771-1

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12771-1

Keywords: CRISPR-Cas9, genome editing, base editing, prime editing, gene delivery, primary cells, genetic disorders, sickle cell disease, β-thalassemia, cystic fibrosis, diabetes mellitus, CRISPR

Amorphophallus is an economically and ornamentally important genus in the arum family, Araceae. Traditional identification is challenging because members of the genus show pronounced morphological plasticity, and the timing of leaf and flower emergence can also be separated. The study therefore used chloroplast-genome comparisons to investigate genome structure, codon usage bias and phylogenetic relationships, including the placement of Amorphophallus among other sampled Araceae.

Researchers sequenced, assembled and annotated chloroplast genomes from A. pendulus, A. glaucophyllus, A. fornicatus, A. macrophyllus, A. titanum and A. hewittii using the PacBio Revio platform. When combined with data from 10 additional species, the dataset covered 16 Amorphophallus taxa. Their chloroplast genomes ranged from 152,492 to 185,810 base pairs and displayed the characteristic quadripartite structure, with the study treating differences in genome size, inverted-repeat regions and gene regions as part of the comparative analysis.

Structural variation was detected in the inverted-repeat regions of several genomes. Expansion of this region occurred in A. macrophyllus and A. glaucophyllus, while contraction was found in A. fornicatus. The comparison also identified the chloroplast gene accD as a highly variable region, with a reported nucleotide diversity value of Pi = 0.1217. These results show that the sampled genomes share a broad structural pattern while differing in specific regions relevant to comparative analysis.

The researchers also assessed codon usage across the 16 chloroplast genomes. Codons showed a bias toward sequences ending in A or U rather than G or C, and the analysis identified natural selection as the predominant driving force behind this pattern. Despite variation among species, six codons were classified as optimal in all 16 Amorphophallus species, indicating a common set of preferred codons within the sampled group. The analysis therefore characterized both shared codon-usage features and differences among individual taxa.

Only C-to-U base-editing events were detected in the chloroplast genomes of the 16 species. In other words, the reported editing pattern was directional within this dataset: cytosine-to-uracil changes were observed, but the analysis did not identify the corresponding U-to-C events. The findings add another layer to the comparison of chloroplast genomes, alongside genome-size variation, inverted-repeat changes and codon-usage bias.

One of the study’s most direct identification results came from its search for an informative DNA barcoding region. The researchers identified the genomic segment spanning 80,401–81,200 base pairs as an optimal barcoding window for the sampled species. Five diagnostic loci within that window could discriminate all 16 Amorphophallus species examined. This finding is specific to the comparative sample used in the study and provides a molecular marker for distinguishing the included taxa.

Phylogenetic reconstruction recovered all sampled Amorphophallus species as a monophyletic group, which the researchers divided into three major clades: Continental Asia I, Continental Asia II and Southeast Asia. The Southeast Asia clade contained A. fornicatus, A. pendulus, A. hewittii and A. titanum. The Continental Asia II clade included A. glaucophyllus, A. macrophyllus, A. konjac, A. krausei, A. albus and A. kachinensis. Continental Asia I comprised A. coaetaneus, A. tonkinensis, A. yunnanensis, A. kiusianus, A. muelleri and one accession that was not identified to species.

Within the wider Araceae sample, Symplocarpus was sister to the remaining sampled members of the family. Amorphophallus was most closely related to Syngonium, Xanthosoma, Zomicarpella and Caladium. Together, the chloroplast analyses provide a comparative account of structural evolution, codon usage and lineage relationships in 16 Amorphophallus species, while the five-locus barcoding window offers a sequence-based means of discriminating the taxa included in the study. The authors identify species identification, germplasm conservation and molecular breeding as areas supported by these findings.

Subject of Research: Comparative chloroplast genomics of structure, codon usage and phylogeny in 16 Amorphophallus species

Article Title: Comparative chloroplast genomics in Amorphophallus: revealing codon usage patterns and phylogenetic clades across 16 species

Article References: Shi, H., Lu, Q., Qi, Y., Yang, M., Zhao, M., Gao, P., Huang, F., Zhao, J., Li, L., & Yu, L. (2026). Comparative chloroplast genomics in Amorphophallus: revealing codon usage patterns and phylogenetic clades across 16 species. BMC Plant Biology. https://doi.org/10.1186/s12870-026-09945-1

Image Credits: AI Generated

DOI: 10.1186/s12870-026-09945-1

Keywords: Amorphophallus, chloroplast genomes, comparative genomics, codon usage bias, plant phylogenetics, DNA barcoding, genome structure, Araceae, base editing, species identification, Comparative, chloroplast

Kohn is a distinguished professor of microbiology, immunology and molecular genetics in the UCLA College and of pediatrics and molecular and medical pharmacology in the David Geffen School of Medicine at UCLA. He is also a member of the UCLA Broad Stem Cell Research Center and the UCLA Health Jonsson Comprehensive Cancer Center. A pediatric bone marrow transplant physician as well as a gene therapy researcher, he has spent nearly 40 years developing and advancing treatments through clinical trials, with the aim of improving care, expanding treatment choices and reaching more patients. “It’s a great recognition of a lifetime of work and research,” Kohn said. “We don’t do this to get awards — we do it because we’re trying to accomplish something for our patients.”

The foundation of Kohn’s career was shaped by several influential mentors. Richard Hong, whose work treating children with severe combined immunodeficiency, drew Kohn into the field, provided his initial inspiration. R. Michael Blaese mentored him during a fellowship at the National Institutes of Health, while Robertson “Robbie” Parkman, his division chief at Children’s Hospital Los Angeles, later guided his professional development. Kohn also credited the research collaborators and clinical colleagues who worked alongside him, describing himself as the conductor in an orchestra of “virtuoso players.” He was recruited to UCLA in 2009 by stem cell and gene therapy leaders Owen Witte, Judith Gasson and James Economou, who he said helped create the interdisciplinary environment and research resources needed for his work to flourish.

One of Kohn’s most consequential contributions is his work on adenosine deaminase-deficient severe combined immunodeficiency, known as ADA-SCID. In this rare disorder, the absence of functional ADA enzyme cripples the immune system by preventing healthy immune cells from developing normally. Kohn and his colleagues treated their first patient with gene therapy in 1993 and have spent more than three decades refining an approach that adds a healthy copy of the ADA gene to a patient’s own blood-forming stem cells. Once modified, those stem cells can generate immune cells capable of restoring immune function. Long-term results published in 2025 from the largest study of the therapy to date found that immune function was restored and maintained in 59 of 62 children. Kohn and his collaborators are now working to move the treatment toward approval by the U.S. Food and Drug Administration so that it can become more widely available.

The rationale for this strategy lies partly in the limitations Kohn observed while performing bone marrow transplants. A conventional transplant can depend on finding a sufficiently well-matched donor, and donor immune cells may attack the recipient’s body, producing graft-versus-host disease. Reusing a patient’s own genetically modified stem cells avoids that donor-recipient mismatch and can require substantially less chemotherapy than a donor transplant. It may also eliminate the need for immune-suppressing medication after treatment. The approach illustrates a central principle of ex vivo gene therapy: a patient’s cells can be removed, genetically corrected in a laboratory and returned to the body to rebuild a deficient system. The technical simplicity of that overview conceals the considerable work involved in delivering genes effectively, preparing patients for therapy and demonstrating that any immune recovery will endure.

That same platform has produced another historic regulatory milestone. Kohn led UCLA’s participation in a multicenter clinical trial of a gene therapy for severe leukocyte adhesion deficiency type I, or LAD-I, a potentially fatal immune disorder that impairs the ability of white blood cells to move from the bloodstream into tissues. In March 2026, the FDA approved the therapy, called Kresladi, after all nine children enrolled in the trial survived and showed sustained restoration of immune function. Kresladi is the first FDA-approved treatment for severe LAD-I and the first gene therapy in which Kohn has been involved to secure regulatory approval. The result also demonstrates how a rare-disease trial can validate multiple parts of a treatment strategy at once: the biological mechanism, the procedure for modifying stem cells, the conditioning regimen required before reinfusion and the durability of the immune correction.

Kohn’s decade-long work on gene therapy for sickle cell disease has yielded additional insights even though the source does not describe that program as having reached approval. His research helped clarify the effects of chemotherapy conditioning, inflammation and gene delivery, knowledge that may improve future treatments for sickle cell disease and related blood disorders. These issues are central to translating a promising laboratory result into a tolerable and effective clinical treatment. Conditioning makes space for corrected cells to take residence in the bone marrow, but it can also provoke inflammation and other adverse effects. Efficient gene delivery is likewise essential for achieving a clinically meaningful level of functional protein. By examining those barriers across diseases, Kohn’s laboratory has helped refine the platform rather than treating each inherited disorder as an entirely separate scientific problem.

The broader laboratory is now extending blood stem cell gene therapy to additional conditions while investigating gene-editing technologies such as CRISPR-Cas9 and base editing. Current projects include potential treatments for alpha thalassemia major, mucopolysaccharidosis type IIIB, also known as MPS IIIB or Sanfilippo syndrome type B, Angelman syndrome and X-linked agammaglobulinemia. Conventional gene addition supplies a functional copy of a gene, whereas editing seeks to alter DNA directly, including correcting a disease-causing mutation or changing a regulatory sequence. These complementary approaches could expand the range of disorders addressable by stem cell therapy. They also raise distinct safety and delivery questions, making the long-term clinical data generated by earlier programs important for evaluating where correction, addition or precise editing offers the most benefit.

Beyond laboratory and clinical research, Kohn has held prominent leadership positions in gene and cell therapy. He has served as president of both the American Society of Gene and Cell Therapy and the Clinical Immunology Society, and as a member and chair of the National Institutes of Health Recombinant DNA Advisory Committee. His previous honors include the American Society of Gene and Cell Therapy’s outstanding achievement award, a career achievement award from the International Society for Cell and Gene Therapy, the Distinguished Clinical Scientist Award from the Doris Duke Charitable Foundation and a lifetime achievement award from the Pediatric Blood & Marrow Transplant Consortium. He has also been recognized for mentoring the next generation of physician-scientists and gene therapy researchers, extending his influence beyond his own laboratory and clinical trials. Kohn said election to the academy is not a reason for him to stop. “This is what I like doing. This is what I’m good at,” he said. “And so I plan to keep doing this, trying to bring new treatments to the clinic for patients.”

Subject of Research: Donald Kohn’s development of blood stem cell gene therapies and election to the National Academy of Medicine

Article Title: UCLA gene therapy pioneer Dr. Donald Kohn elected to National Academy of Medicine

Article References: UCLA gene therapy pioneer Dr. Donald Kohn elected to National Academy of Medicine. Original publication (Bibliographic details unavailable.)

Image Credits: AI Generated

DOI: Not provided

Keywords: Donald Kohn, gene therapy, blood stem cells, ADA-SCID, severe LAD-I, Kresladi, sickle cell disease, CRISPR-Cas9, base editing, UCLA, National Academy of Medicine, rare immune disorders

Most plant cytidine base editors are built as one continuous protein. A catalytically weakened version of Cas9, known as SpCas9 nickase, is fused to a cytidine deaminase enzyme. This arrangement keeps the enzyme close to the single-stranded DNA exposed when the guide RNA directs the nickase to its target. The strategy is generally powerful, but prolonged contact can allow the deaminase to modify several nearby bases, create unwanted DNA-repair outcomes or contribute to small insertions and deletions, or indels. Testing a new deaminase, linker or Cas9 variant can also require rebuilding and reconstituting the entire fusion protein, a laborious process in plants because transformation is slow.

Free-BE takes a deliberately simple approach to this design problem. The nickase and CDA1 are encoded on separate plasmids and expressed independently, without an intein, engineered protein interaction or RNA-based recruitment system. The central hypothesis was that the two proteins could still work together if they encountered the same target temporarily. If the deaminase were not tethered continuously to the nickase, it might spend less time exposed to the target DNA. That shorter exposure could preserve useful C-to-T conversions while reducing collateral editing. Researchers compared this modular configuration, called Fused-BE, with a conventional nCas9–CDA1 fusion in the moss APT reporter system, where mutations that inactivate the APT gene confer resistance to the toxic adenine analogue 2-fluoroadenine.

In moss, both systems edited APT, but the fused version was clearly more productive. With one guide RNA, Fused-BE inactivated APT in 7.1% of regenerating plants, compared with 2.9% for Free-BE; with the second guide, the rates were 5.6% and 1.9%, respectively. Expression of the nickase alone produced negligible mutation frequencies of 0.004% and 0.008%, showing that most of the activity attributed to Free-BE came from its deaminase component. The lower yield was therefore a real limitation, but it did not indicate that independent expression prevents the enzyme from functioning at a guide-defined site.

The quality of the resulting edits differed as strikingly as their frequency. With the first guide, 52.8% of the mutations produced by Fused-BE contained indels, versus 7.1% with Free-BE—a roughly sevenfold reduction that was not merely a consequence of the system’s lower overall efficiency. Free-BE also produced a higher proportion of plants carrying only one edited cytidine: 55% with the first guide and 82% with the second, compared with 6% and 49% for the fused editor. The two architectures did not generate identical molecular outcomes, however. Free-BE produced fewer canonical C-to-T transitions and relatively more transversions, especially C-to-G, suggesting that changes in the timing and proximity of the deaminase may influence how the resulting U:G mismatch is repaired.

Targeted safety checks did not reveal unintended mutations in the candidate off-target sites examined in moss. The researchers identified four potential off-target loci for one guide using a sequence-similarity score and sequenced them in plants edited with either system. No mutations were detected at these sites in the tested lines. That finding is encouraging but limited: the analysis covered only a few predicted genomic locations for a single guide and did not measure genome-wide off-target activity, RNA editing or off-target effects in maize and wheat. The result therefore supports the need for broader specificity studies rather than establishing genome-wide specificity for Free-BE.

Free-BE then moved into a crop-based reporter test in maize. Researchers engineered a nonfunctional blue fluorescent protein so that one C-to-T edit would restore a functional green fluorescent protein. Maize protoplasts receiving the fused editor converted 1.5% of reporter molecules, while those receiving Free-BE achieved 0.6% conversion. Negative controls produced almost no GFP-positive cells. The experiment demonstrated that unlinked components can generate targeted base editing in maize cells, but because the assay measured correction of a transient reporter rather than editing of an endogenous maize gene, stable maize experiments are still needed to establish how the system performs in the crop genome.

Wheat provided the most demanding validation, involving Agrobacterium-mediated transformation of the Fielder cultivar and two guide RNAs directed at TaGASR7 and TaLOX2. Each guide perfectly matched three homoeologous copies, giving six possible endogenous targets for multiplex editing. Of 291 transformed T0 plantlets carrying Fused-BE, 235 contained an edited cytidine at one or more targets, an editing rate of 80.8%. Free-BE edited at least one target in 33 of 77 transformants, or 42.9%. Both systems could multiplex several loci: 9.1% of edited Free-BE plants carried changes in five of the six target genes, while 11.5% of edited Fused-BE plants showed changes in all six. The conventional editor therefore remained more efficient, but the modular system retained considerable activity and generated combinations of edits across multiple wheat homoeologs.

The wheat sequence data also reproduced Free-BE’s cleaner editing profile. At the TaLOX2 target, which contains seven cytidines in the guide’s spacer, 50% of Free-BE-edited plants carried one edited cytidine, compared with 21.7% for Fused-BE. Dual and triple edits were correspondingly less frequent with Free-BE, occurring in 10.0% and 3.8% of plants rather than 22.6% and 12.6%. Fused-BE also generated occasional edits near the guide’s PAM-proximal edge and outside the spacer region, whereas Free-BE did not show those events in the analyzed wheat plants. Unlike in moss, Free-BE did not consistently increase C-to-G or C-to-A transversions in wheat, indicating that the effect of separating the editor components may depend on the plant species, target sequence and biological context.

Overall, the study presents Free-BE as a precision-first alternative to the speed and efficiency of fused base editing. Its principal advantages are fewer indels, a greater likelihood of isolated single-cytosine changes, no detected edits beyond the predicted window in the wheat dataset, and a modular design that allows deaminases, Cas9 variants and expression strategies to be tested without repeatedly constructing a complete fusion. Its disadvantages are equally clear: editing frequencies were consistently lower, and the spectrum of nucleotide substitutions could be less predictable in moss. Optimizing the expression levels and timing of the two components may improve performance, while stable maize tests and comprehensive DNA and RNA off-target analyses will be needed to define the system’s full utility. For precision breeding, the results point to a promising bargain: a lower chance of producing the desired allele may be acceptable if it comes with a lower chance of altering the genetic neighborhood around it.

Subject of Research: Evaluation of a modular cytidine base-editing strategy in moss, maize, and wheat

Article Title: Validation of a Modular Base Editing Strategy, Free‐BE, in Physcomitrium patens, Maize, and Wheat

Article References: Guyon‐Debast, A., Javelle, M., Perroud, P.-F., Pichon, J.-P., Paul, W., & Nogué, F. (2026). Validation of a Modular Base Editing Strategy, Free‐BE, in Physcomitrium patens , Maize, and Wheat. Plant Direct, 10(10), Article e70196. https://doi.org/10.1002/pld3.70196

Image Credits: AI Generated

DOI: 10.1002/pld3.70196

Keywords: base editing, CRISPR, genome editing, cytidine deaminase, wheat, maize, moss, plant breeding, DNA repair, precision breeding, Validation, Modular

The review, led by Samruddhi Kaknale and Varnitha Manikantan of Symbiosis Medical College for Women in Pune, India, together with collaborators at Symbiosis School of Biological Sciences, Karunya Institute of Technology and Sciences, and Manipal Institute of Technology Bengaluru, systematically maps the pathobiology of infant neuroblastoma, the mechanics of CRISPR/Cas9 targeting, and the nanocarrier platforms that might deliver the editing machinery safely in vivo. The authors are careful to frame the work as an investigational hypothesis rather than an established clinical application, noting that direct evidence in infant neuroblastoma remains limited and that most findings come from general neuroblastoma models, non-cancer models, or adult cancers that should not be interpreted as proof of efficacy in infants.

At the heart of the therapeutic concept lie two molecular hallmarks: MYCN amplification and activating ALK mutations. MYCN is a potent transcription factor that drives proliferation, suppresses apoptosis, and propels tumor progression, and its amplification is an established adverse prognostic feature associated with advanced stage, rapid growth, and poor outcome. ALK mutations, meanwhile, produce constitutive kinase activity that fuels oncogenic signaling. Because these alterations define the biology of high-risk disease, they represent the most attractive targets for precision gene editing, offering a shift from broad cytotoxic chemotherapy toward highly selective molecular intervention.

The CRISPR/Cas9 system itself is elegantly simple in principle. It consists of the Cas9 nuclease and a single guide RNA, a synthetic chimeric RNA containing a scaffold for Cas9 binding and a 20-nucleotide spacer complementary to the target DNA. For the commonly used Streptococcus pyogenes Cas9, target recognition requires a 5′-NGG-3′ protospacer-adjacent motif. Once directed to its genomic address, Cas9 induces a targeted double-strand break that the cell repairs through endogenous mechanisms. The predominant pathway, non-homologous end joining, is error-prone and typically generates small insertions or deletions that frameshift and inactivate the target gene, a powerful strategy for knocking out oncogenes such as MYCN or ALK. The less frequent homology-directed repair pathway uses a homologous DNA template and can, in principle, correct specific point mutations, such as activating ALK variants, with meticulous precision.

Recent advances have refined this toolkit further. Base editors harness a catalytically impaired Cas9 fused to a deaminase to convert single nucleotides directly, for example C to T or A to G, without double-strand breaks. Prime editors combine nCas9 with reverse transcriptase and a prime editing guide RNA to direct templated insertions, deletions, and all twelve possible base-to-base conversions, offering search-and-replace capabilities that reduce the risk of indels and chromosomal rearrangements. The review notes that direct evidence for base or prime editing in neuroblastoma remains limited, marking these approaches as avenues for future investigation rather than validated options.

Preclinical evidence, however, is already compelling in cell lines and animal models. High-throughput CRISPR screens have identified genetic dependencies in neuroblastoma, including a reliance on the PRC2 components EZH2, EED, and SUZ12 in MYCN-amplified models. Direct knockout of MYCN and ALK in various neuroblastoma cell lines has produced significant reductions in cell proliferation, inhibition of tumor growth in vitro and in vivo, and induction of apoptosis. CRISPR interference targeting the MYCN promoter has effectively repressed MYCN expression and suppressed neuroblastoma cell growth, while knockout of uPAR, a gene linked to cellular invasion, has inhibited invasive phenotypes, suggesting the technology can also blunt metastasis by targeting genes involved in tumor cell motility.

Yet the review is unflinching about the obstacles. Delivering Cas9 and its guide RNA into a sufficient percentage of tumor cells in vivo remains the central challenge, complicated by the large size, negative charge, immunogenicity, and degradation susceptibility of CRISPR cargoes. The Cas9 protein, derived from bacteria, can elicit innate and adaptive immune responses that clear edited cells or render subsequent treatments unproductive, and preexisting adaptive immunity to Cas9 proteins has been identified in humans. Off-target editing persists despite improvements in guide RNA design and high-fidelity Cas9 variants, and achieving uniform editing efficiency across a heterogeneous solid tumor microenvironment, with poor penetration and variable cellular uptake, is extraordinarily difficult. For infants, whose expected lifespans stretch across decades, persistent Cas9 activity, potential germline exposure, chromosomal rearrangements, large deletions, structural variants, and long-term effects in normal tissues demand careful assessment and lifelong follow-up before any clinical translation.

Nanoparticle delivery systems emerge as the leading candidates to surmount these barriers. Lipid nanoparticles, the most clinically advanced platform and the technology behind successful mRNA vaccines, typically comprise ionizable lipids, helper lipids like cholesterol and phospholipids, and PEGylated lipids. Ionizable lipids are positively charged at acidic pH but neutral at physiological pH, facilitating nucleic acid complexing and endosomal escape after cellular uptake, while PEGylated lipids preserve systemic circulation by preventing rapid elimination by the reticuloendothelial system. Delivery of Cas9 mRNA with sgRNA allows transient protein expression, minimizing off-target effects and immune activation associated with prolonged Cas9 activity, whereas pre-formed Cas9 ribonucleoproteins offer reduced immunogenicity and faster editing onset but are more complicated to encapsulate due to their size.

Polymeric and inorganic nanocarriers expand the design space. Biodegradable polymers such as PLGA, PEG, chitosan, and dendrimers can encapsulate CRISPR components with adjustable degradation and sustained release, and PLGA’s capacity to safeguard sensitive payloads makes it widely used for surface functionalisation aimed at neuroblastoma cell targeting. Gold nanoparticles are biocompatible and easily functionalized for direct conjugation with Cas9 and sgRNA, with photothermal applications, while magnetic nanoparticles enable magnetic targeting and MRI tracking, and mesoporous silica nanoparticles provide high loading, controlled release, and rigid protection of CRISPR cargo. For the minority of patients with CNS involvement or relapse, blood-brain barrier penetration strategies, including receptor-mediated transcytosis via ligands binding the transferrin, insulin, or low-density lipoprotein receptors, cell-penetrating peptides like TAT and R8, and focused ultrasound combined with microbubbles to transiently and locally disrupt the barrier, are being explored, though the review cautions that enhanced permeability does not necessarily indicate efficient nanoparticle delivery to the brain and may increase the risk of unintended CNS exposure and neurotoxicity.

The authors conclude that robust preclinical validation in relevant in vitro and in vivo models, including organoids, patient-derived xenografts, and orthotopic systems that precisely mimic the human disease, is essential for determining efficacy, characterizing biodistribution, evaluating off-target effects, and establishing a preliminary safety profile. Ethical and regulatory considerations add further complexity, as infants lack capacity for informed consent, requiring parents or legal guardians to decide in the child’s best interests amid the potentially irreversible nature of genomic modification. Future work should prioritize more selective nanoparticles, gene-editing tools with high precision and minimal off-target effects, and internationally harmonised regulatory frameworks for paediatric applications. With sustained interdisciplinary collaboration, the convergence of nanoparticle delivery and CRISPR/Cas9 precision may ultimately enable safer and more effective therapies for infants with neuroblastoma, though the review emphasizes that current evidence remains predominantly preclinical and is not yet sufficient to establish the optimal nanoparticle or CRISPR technique for infant neuroblastoma.

Subject of Research: Nanoparticle-enabled CRISPR/Cas9 gene editing as a potential therapy for infant neuroblastoma

Article Title: Precision gene editing in infant neuroblastoma: a review of nanoparticle: enabled CRISPR/Cas9 therapies

Article References: Kaknale, S., Ranjan, A., Enoch, I. V. M. V., Vyas, A., & Manikantan, V. (2026). Precision gene editing in infant neuroblastoma: a review of nanoparticle: enabled CRISPR/Cas9 therapies. Molecular Biology Reports, 53(1), Article 1725. https://doi.org/10.1007/s11033-026-12916-2

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12916-2

Keywords: infant neuroblastoma, CRISPR/Cas9, nanoparticles, lipid nanoparticles, gene therapy, MYCN, ALK, drug delivery, paediatric oncology, base editing, prime editing, preclinical models

The scientific foundation for this transformation was laid in 1995, when a landmark study identified SMN1 as the disease-causing gene. The authors found that SMN1 was deleted in 98.7 percent of patients in a 229-person cohort and mutated in the remainder. Crucially, humans carry a nearly identical backup copy called SMN2, which differs by a single base-pair transition from C to T in exon seven. That single nucleotide inactivates an exonic splicing enhancer and introduces an exonic splicing silencer, causing roughly 90 percent of the protein produced from SMN2 to be truncated and degraded by cellular machinery. Because SMA severity is inversely related to the number of SMN2 copies a patient carries, this backup gene became the central target for drug development.

Three FDA-approved disease-modifying therapies now anchor the treatment landscape. Nusinersen, marketed as Spinraza, is an antisense oligonucleotide that binds to a specific site within SMN2 pre-mRNA to promote inclusion of exon seven, restoring production of full-length SMN protein. Its development traced back to the discovery of an intronic splicing silencer called ISS-N1, and animal studies showed that blocking this element dramatically increased exon seven inclusion. The pivotal Phase 3 ENDEAR trial demonstrated significant improvement in infants with SMA Type 1, leading to accelerated FDA approval in 2016. Notably, in early 2026 the FDA approved a higher-dose regimen based on the DEVOTE study, with a safety profile consistent with more than a decade of clinical data.

Onasemnogene abeparvovec, known commercially as Zolgensma, takes a fundamentally different approach. It is a non-replicating adeno-associated virus serotype 9 vector that delivers a full-length SMN1 gene under the control of a hybrid promoter, capable of crossing the blood-brain barrier to reach spinal motor neurons after a single intravenous injection. A meta-analysis conducted four years after the drug’s release found mostly positive results, particularly in patients treated early in life, with motor milestone improvements that in many studies exceeded those achieved with nusinersen. A biodistribution study of post-mortem patient tissue confirmed vector genome presence throughout the central nervous system and in peripheral organs including skeletal muscle, heart, lung, and stomach.

Risdiplam, sold as Evrysdi, is the most recent addition and the first orally administered small-molecule SMN2 splicing modifier. The compound stabilizes the interaction between the 5-prime splice site on exon seven and the U1 small nuclear ribonucleoprotein, strengthening the splice site and reducing degradation. Unlike nusinersen, risdiplam crosses the blood-brain barrier because it does not bind to multi-drug resistant protein 1 and therefore cannot be pumped out of the central nervous system. A striking case study even documented prenatal therapy, with the drug administered orally to a mother carrying a confirmed SMA Type 1 fetus beginning at 32 weeks and 5 days of gestation; at 30 months of age, the child showed no manifestations of the disease. In a matching-adjusted indirect comparison, risdiplam-treated Type 1 patients showed a 78 percent reduction in mortality relative to historical nusinersen cohorts.

Despite these remarkable gains, the review emphasizes that the therapies are not curative. In 2024, approximately 75 percent of patients in the CureSMA database had received at least one disease-modifying therapy, yet only 47 percent of children and 19 percent of adults with SMA are ambulatory. Since the dawn of the therapy era, SMA mortality has dropped by nearly 80 percent, but 36 percent of patients still require some form of breathing support, and 89 percent of adults report that reaching new motor milestones remains their greatest unmet need. As patients live longer, a constellation of comorbidities has come into sharper focus, reshaping the clinical agenda.

Orthopedic complications illustrate this shift vividly. Between 60 and 90 percent of children with SMA develop scoliosis, which can complicate the intrathecal injections required for nusinersen and, if untreated, lead to chest deformities that further impair pulmonary function. Respiratory failure remains the leading cause of death, implicated in 72 percent of known SMA fatalities in a recent review. Gastrointestinal problems, including chronic constipation, dysphagia, and gastroesophageal reflux, are common, and eating difficulties can produce metabolic abnormalities such as glucose intolerance and insulin resistance. Cardiac irregularities, though less frequent, have been documented in infantile patients, ranging from congenital heart defects to cardiomyopathy.

The review also delves deeply into why motor neurons are selectively vulnerable to SMN deficiency, a question that remains only partially answered. SMN protein is best known for assembling small nuclear ribonucleoproteins, the complexes essential for recognizing splice sites and removing introns from pre-mRNA. Motor neurons harbor disproportionately high numbers of Cajal bodies, the nuclear structures where snRNP maturation is completed, likely reflecting their extraordinary demand for RNA metabolism to sustain constant electrical signaling. Low SMN reduces Cajal body numbers and, by extension, ribosome production. SMN also interacts with mRNA-binding proteins such as HuD, IMP1, and beta-actin that govern axonal transport and growth, and its deficiency disrupts neuromuscular junction maturation before symptoms even appear.

Looking forward, the authors survey a rich pipeline of alternative strategies. Induced pluripotent stem cells derived from patient skin fibroblasts have become powerful tools for drug screening and disease modeling, revealing pathways such as Fas ligand-mediated apoptosis and calcium dysregulation in cardiomyocytes. Gene editing approaches are particularly striking: a one-time base editing technique achieved an average of 87 percent permanent conversion of the disease-causing point mutation in mouse models with no detected off-target effects, while CRISPR-based prime editing of the ISS-N1 silencer sequence restored full-length SMN2 production in patient cells. Epidural spinal cord stimulation in a small cohort of adults produced a 40 percent increase in mean step length over four weeks, with benefits persisting even when the stimulator was switched off.

The review closes with a call for combination treatment strategies that build on existing therapies while addressing residual disease burden. Investigational agents such as apitegromab, a monoclonal antibody that blocks myostatin’s inhibition of skeletal muscle growth, are already being tested alongside approved drugs. The authors stress that SMA is fundamentally a multi-organ disease requiring multifactorial management, and that future research must ensure patients of all ages, including the growing population of adults living with the disease, are represented in treatment development. What began as a uniformly fatal diagnosis has become a chronic, manageable condition for many, but converting survival into truly restored function remains the defining challenge of the next decade.

Subject of Research: Disease-modifying therapies and future treatment directions for spinal muscular atrophy

Article Title: Spinal muscular atrophy in the disease-modifying therapy era: successes, limitations and future directions

Article References: Sexton, M. M., Crow, E. W., & Peng, C. A. (2026). Spinal muscular atrophy in the disease-modifying therapy era: successes, limitations and future directions. Frontiers in Molecular Medicine, 6, Article 1901311. https://doi.org/10.3389/fmmed.2026.1901311

Image Credits: AI Generated

DOI: 10.3389/fmmed.2026.1901311

Keywords: spinal muscular atrophy, SMN1, SMN2, nusinersen, onasemnogene abeparvovec, risdiplam, gene therapy, motor neurons, antisense oligonucleotide, comorbidities, base editing, iPSCs

Duchenne muscular dystrophy is an X-linked disorder caused by mutations in the DMD gene, the largest gene in the human genome, spanning 79 exons. These mutations abolish production of dystrophin, a structural protein that acts as a molecular shock absorber, linking the internal cytoskeleton of muscle fibers to the surrounding extracellular matrix. Without it, muscle cells degenerate under the mechanical stress of contraction, leading to progressive wasting, loss of ambulation, cardiomyopathy and premature death. The gene’s sheer size and its array of alternative promoters give rise to multiple dystrophin isoforms of different lengths: the full-length Dp427m isoform dominates in skeletal and cardiac muscle, while shorter versions such as Dp260 in the retina, Dp140 in the central nervous system and kidney, Dp116 in Schwann cells, and the ubiquitous Dp71 and Dp40 serve specialized roles in non-muscle tissues. Where a patient’s mutation falls on the gene determines which isoforms are lost, and thus which tissues bear the brunt of the disease.

Existing preclinical models fall short of capturing this complexity. The classic mdx mouse carries a nonsense mutation in the murine Dmd gene, but mouse and human DMD differ substantially in sequence, exon–intron organization and splicing regulation, and the mouse phenotype is comparatively mild. Humanized mice, in which specific murine exons have been replaced with human sequences, allow editing tools designed against human targets to be tested in vivo — but each line models only a single mutation. Because Duchenne lacks mutational hot spots and instead displays extreme heterogeneity across the gene, building a bespoke mouse for every clinically relevant variant is impractical, particularly for rare mutations. The Korean team, led by Hyuk-Jin Cha and Hyukjin Lee, reasoned that a renewable, patient-derived tissue that carries the patient’s own mutation might sidestep this limitation entirely.

Their solution exploits a structure usually regarded as a laboratory nuisance. When human pluripotent stem cells are injected into immunodeficient mice, they form teratomas containing disorganized derivatives of all three germ layers, including skeletal, cardiac and smooth muscle. Teratomas derived from Duchenne patient iPS cells recapitulate the absence of dystrophin, effectively recreating a patch of dystrophic human tissue inside a mouse. The researchers drew an analogy to patient-derived tumor xenografts, a mainstay of oncology drug development, and adapted the same framework to neuromuscular disease. From a bank of nine patient-derived iPS lines carrying missense, nonsense, duplication and deletion mutations, they focused on a line harboring a C-to-T transition in exon 6 that creates a premature stop codon — a mutation directly correctable by an adenine base editor, which chemically converts A·T base pairs to G·C without cutting the DNA double helix.

First, the team built an isogenic pair. Using electroporation, they delivered the adenine base editor into the mutant iPS cells and isolated edited clones that retained full pluripotency, confirmed by marker gene expression, alkaline phosphatase staining and teratoma formation. When differentiated in a dish over 16 days, the edited cells matured into spontaneously contracting cardiomyocyte-like cells, and immunoblotting revealed that the full-length Dp427m protein, absent in the mutant line, had been restored. A stepwise myogenic protocol — guiding cells through paraxial mesoderm, myogenic progenitor and myoblast stages over roughly 90 days — produced multinucleated, striated myotubes that likewise expressed robust Dp427m. These experiments established that correcting the single exon 6 mutation was sufficient to rescue the muscle-specific isoform in both cardiac and skeletal muscle lineages in vitro.

The ex vivo test followed. The researchers generated day-12 myogenic progenitors from the edited iPS cells, confirmed their expression of the progenitor markers PAX7 and MyoD by flow cytometry and immunofluorescence, and injected 100,000 of these cells into dystrophic teratomas grown from the uncorrected mutant cells. Four weeks later, immunoblotting showed that the engrafted progenitors had restored several shorter dystrophin isoforms — bands at approximately 117, 71 and 55 kilodaltons, presumably corresponding to Dp116, Dp71 and Dp40 — within the teratoma environment. Notably, however, neither the edited teratomas nor the progenitor-engrafted ones displayed the full-length Dp427m band seen in mature mouse muscle, suggesting that the teratoma’s developmental context did not yet support the maturation level required for the full-length protein.

The in vivo arm of the study tackled a delivery problem that has dogged the base-editing field. Adenine base editors such as ABE8e span roughly 5.4 kilobases, exceeding the approximately 4.7-kilobase packaging limit of adeno-associated virus vectors — and AAV immunogenicity has already produced severe, sometimes fatal adverse events in Duchenne gene therapy trials. Messenger RNA encapsulated in lipid nanoparticles, the platform proven by COVID-19 vaccines, accommodates large transcripts readily and avoids viral immunogenicity. The team first attempted a split-editor strategy, encoding the editor in two fragments joined by intein sequences that self-splice after translation, but found that the split mRNAs produced essentially no protein, likely because their engineered 5′ untranslated regions impaired ribosomal scanning. Full-length constructs with naturally functional leader sequences, by contrast, expressed robustly. Formulated by microfluidic mixing into uniform spherical nanoparticles of roughly 60 to 76 nanometers with low polydispersity, the SpRY mRNA–LNP conjugates outperformed even plasmid delivery when tested in patient iPS cells.

Injected directly into teratomas in mice, the editor showed a clear dose response: a single 10-microgram dose yielded about 1 percent editing, while escalating to 80 micrograms achieved up to roughly 15 percent in primary teratomas. Because teratomas vary unpredictably in size, composition and maturation, the researchers then devised a secondary transplantation scheme — sectioning one primary teratoma into four pieces and regrowing them in additional mice — to produce more uniform tissue. In these sequentially matured teratomas, editing efficiencies approached 100 percent at the highest dose, and, strikingly, two of four high-dose samples showed robust restoration of full-length Dp427m by long-exposure immunoblotting. Targeted sequencing of six predicted off-target exonic sites revealed no detectable A-to-G conversion above background, even at the highest dose, underscoring the specificity of the approach in human tissue.

Why did only some fully edited teratomas produce the full-length protein? Single-cell RNA sequencing of a Dp427m-positive and a Dp427m-negative teratoma provided the answer. Both contained broadly distributed DMD transcripts reflecting baseline activity of shorter isoforms, but only the positive sample harbored an enriched myogenic cluster — one of twenty identified cell populations — marked by elevated expression of skeletal muscle genes such as RYR3, LDB3, FBXO32 and MYOM1, along with cardiac maturation markers including TNNT2 and RYR2. Myogenic maturation, the data showed, is a critical determinant of whether corrected DNA translates into the clinically relevant full-length isoform. The authors caution that teratoma growth is inherently difficult to control and that animal welfare limits restrict teratoma size, so reproducible modeling of Dp427m recovery will require further optimization — potentially through inducible myogenic programming to bias teratomas toward muscle. Still, the platform establishes a proof of concept: a scalable, mutation-inclusive, patient-specific human tissue in which cell therapy and in vivo base editing can be tested side by side, bringing the field a step closer to evaluating personalized gene correction before it is ever infused into a patient.

Subject of Research: A patient-derived teratoma platform for evaluating ex vivo cell therapy and in vivo base editing to restore dystrophin in Duchenne muscular dystrophy

Article Title: Patient-derived teratomas as a humanized platform for dystrophin restoration by ex vivo cell therapy and in vivo base editing

Article References: Park, M. J., Kim, H.-B., Hwang, Y., Kim, Y.-J., Park, J.-C., Lim, H.-K., Park, Y.-G., Moon, S., Lee, H., & Cha, H.-J. (2026). Patient-derived teratomas as a humanized platform for dystrophin restoration by ex vivo cell therapy and in vivo base editing. Experimental & Molecular Medicine. https://doi.org/10.1038/s12276-026-01854-5

Image Credits: AI Generated

DOI: 10.1038/s12276-026-01854-5

Keywords: Duchenne muscular dystrophy, dystrophin, base editing, induced pluripotent stem cells, teratoma, lipid nanoparticles, gene therapy, myogenic progenitors, Dp427m, xenograft model, mRNA delivery, single-cell RNA sequencing

α-thalassemia arises when the α-globin genes, which encode one of the two protein chains of adult hemoglobin, are deleted or defective. Healthy people carry four copies of the α-globin gene, two on each chromosome 16. Carriers with three or two functional copies are usually asymptomatic, and the carrier alleles are extraordinarily common in malaria-endemic regions, reaching frequencies of up to roughly 70 percent because they confer protection against the parasite. But when these alleles combine, the consequences escalate. People inheriting one Southeast Asian deletion allele and one α-globin deletion develop hemoglobin H disease, in which excess β-globin chains form useless β4 tetramers, and about 10 percent of these patients become transfusion dependent. The most devastating outcome occurs in fetuses that inherit no functional α-globin genes at all, a condition known as hemoglobin Bart’s hydrops fetalis syndrome, or BHFS, which normally causes death in utero during the second or third trimester.

Current options for the most severe patients are limited. Hematopoietic stem cell transplantation is the only curative treatment, but it requires a human leukocyte antigen-matched donor, and even when a donor is available, graft-versus-host disease and transplant-related mortality remain real risks. A small but growing number of BHFS survivors, salvaged by intrauterine transfusions and intensive neonatal care, face lifelong transfusion dependence and iron chelation. This therapeutic gap is what drew the Oxford team to an unusual idea: rather than trying to replace the missing α-globin gene, why not switch back on the embryonic gene that once did its job?

The logic rests on a quirk of human development. The α-globin locus on chromosome 16 contains, in sequence, the enhancers R1 through R4, the embryonic ζ-globin gene HBZ, and the two fetal and adult α-globin genes HBA2 and HBA1. During the first, primitive wave of red blood cell formation, ζ-globin is fully expressed and assembles into functional hemoglobin tetramers, including Hb Portland 1 (ζ2γ2) and Hb Portland 2 (ζ2β2), which can substitute for fetal and adult hemoglobins. But by six to seven weeks of gestation, as definitive hematopoiesis takes over, ζ-globin is completely silenced and remains off for the rest of life. Crucially, the most common disease-causing allele, the Southeast Asian deletion, removes both α-globin genes while leaving the enhancers and the ζ-globin gene intact, meaning that in many patients the dormant embryonic gene is still sitting there, waiting to be unlocked.

To find the off switch, the researchers first built an elegant mouse model called DZIA, in which the 4.4-kilobase segment containing the ζ-globin gene was replaced with a modified α-globin gene carrying twenty silent single-nucleotide polymorphisms that allowed its transcripts to be distinguished from endogenous ones. The result was striking: the substituted gene, placed in exactly the position of the embryonic gene, was fully transcribed in definitive erythroid cells, accounting for 40 to 50 percent of total α-like globin output, roughly equivalent to the combined output of two endogenous α-globin genes. This proved that the silencing machinery is encoded locally within that 4.4-kilobase region, not in distant flanking sequences or in the gene’s position within the chromosome’s three-dimensional architecture.

Next came a CRISPR-Cas9 saturation mutagenesis screen across the ζ-globin promoter. Using a reporter mouse in which ζ-globin expression is marked by a fluorescent protein, and refining the search in an immortalized mouse proerythroblast line, the team tiled 47 guide RNAs across 700 base pairs upstream of the transcription start site. The screen converged on two discrete repressive elements: predicted binding motifs for the transcription factors BCL11A and ZBTB7A, also known as LRF. Disrupting the BCL11A motif alone derepressed ζ-globin to about 4 percent of total α-like globin, the LRF motif alone to about 18 percent, and combined disruption, introduced with a cytosine base editor, pushed expression to roughly 26 percent, marginally more than the sum of the individual effects.

The team then built a mouse model carrying these promoter edits, termed ZPE, and crossed it with a new BHFS model that precisely replicates the human Southeast Asian deletion, including its flanking sequences. Untreated homozygous deletion embryos, which produce no α-globin at all, develop normally until embryonic day 15.5, when the last primitive, ζ-globin-expressing red cells disappear from circulation. Thereafter they become pale and growth restricted, develop edema, and by day 17.5 suffer severe hemolytic anemia with large numbers of immature, nucleated erythroblasts churning out nonfunctional β4 tetramers. Embryos carrying the promoter edits on top of the deletion showed dramatic improvement at day 17.5, with near-normal enucleated red blood cells expressing about 7.5 percent ζ-globin mRNA and producing detectable Hb Portland 2. The embryonic protein was, in effect, standing in for the missing adult one.

The human work followed a parallel path. In the HUDEP-2 definitive erythroid cell line, the researchers found that the architecture of the ζ-globin promoter differs subtly from the mouse: two consensus BCL11A motifs lie 148 base pairs apart, with the proximal one overlapping the essential CCAAT box, so editing that site is ineffective. The distal BCL11A site and the LRF site, however, proved to be the productive targets. Base editing both sites raised ζ-globin to about 15 percent of total α-like globin, comparable to levels achieved by knocking out both repressor genes entirely, while leaving erythroid differentiation untouched. Mechanistically, the authors propose a competition model: LRF binding, just 18 base pairs from the CCAAT box, blocks NF-Y from opening the chromatin, and together with BCL11A it prevents the master activator GATA1 from engaging the promoter. When both repressive motifs are mutated, GATA1 newly binds and the gene fires.

The most clinically resonant experiments used primary human CD34-positive stem cells from healthy donors, patients with hemoglobin H disease and a 21-week BHFS fetus. Adenine and cytosine base editors achieved efficiencies of 25 to 90 percent at the two sites, and edited cells differentiated normally. After normalizing for editing efficiency, the ratio of α-like to β-like globin in the hemoglobin H patient samples rose from 24.8 percent, a dangerously low level, to 48.4 percent, with ζ-globin contributing about half of the α-like output. In the BHFS fetal cells, the ratio climbed from 17.1 percent to 50.5 percent. The authors argue that restoring this ratio to around 0.5 would likely be sufficient to rescue the severe α-thalassemia phenotype, and HPLC analysis of edited BHFS cells showed a corresponding reduction in the pathological β4 tetramers that cause the disease.

Caution remains appropriate. Hb Portland 2 has weaker subunit interactions and higher oxygen affinity than adult hemoglobin, and its physiological behavior in adult life must be characterized before clinical application. Editing efficiencies in primary stem cells vary, off-target effects and long-term engraftment have yet to be rigorously assessed, and the authors note that residual low-level ζ-globin expression seen in human carriers appears to involve human-specific mechanisms absent from the mouse. Still, the study establishes a clear road map: a compact, locally acting cis-regulatory module enforces embryonic gene silencing, and precise base editing can dismantle it. Beyond α-thalassemia, the work reinforces an emerging paradigm, already validated by fetal hemoglobin induction in sickle cell disease and β-thalassemia, that developmentally silenced paralogs can be unlocked to compensate for adult gene defects, offering a potentially generalizable route to autologous genome-editing therapies for severe inherited disorders that today demand lifelong transfusion or a matched donor.

Subject of Research: Reactivation of the embryonic ζ-globin gene by promoter base editing as a therapeutic strategy for severe α-thalassemia

Article Title: Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia

Article References: Liu, S., Ejaz, A., King, A. J., Olijnik, A.-A., Sloane-Stanley, J., Scott, C., Amid, A., Biggs, D., Davies, B., Eglinton, J., Martyn, G., Crossley, M., Babbs, C., Kassouf, M. T., & Higgs, D. R. (2026). Reactivation of the embryonic ζ-globin gene ameliorates severe forms of α-thalassemia. Nature Genetics, 58(10), 2619-2629. https://doi.org/10.1038/s41588-026-02770-0

Image Credits: AI Generated

DOI: 10.1038/s41588-026-02770-0

Keywords: α-thalassemia, ζ-globin, gene editing, base editing, BCL11A, ZBTB7A, hemoglobin, erythropoiesis, hydrops fetalis, CRISPR, globin gene regulation, hemoglobinopathies

The technical backdrop to their argument is a field advancing on multiple fronts simultaneously. A review by Pan and colleagues, published in the same journal, traces how CRISPR-Cas9 and its increasingly refined descendants, including base editing, prime editing, and AI-guided RNA design, are converging with cell and vector engineering to offer potentially curative options for children with rare monogenic diseases. Base editing chemically converts one DNA letter into another without cutting both strands of the double helix, while prime editing allows more precise insertions and deletions with fewer unwanted byproducts. Machine learning tools are accelerating the design of RNA molecules and delivery vehicles, shortening development timelines that once stretched over decades.

The clinical results are no longer theoretical. Several gene therapy products now carry approval from the Food and Drug Administration or the European Medicines Agency for pediatric indications, and dozens more sit in active clinical trials. Diseases once uniformly fatal or progressively disabling, such as spinal muscular atrophy and metachromatic leukodystrophy, now have potentially curative options. The commentary describes this as an inflection point, but one whose promise is fragile, because access for children today and development of new therapies tomorrow hinge on coverage decisions made in insurance boardrooms as much as in laboratories.

Price is the most visible obstacle. Atidarsagene autotemcel, marketed as Lenmeldy, is a one-time therapy for metachromatic leukodystrophy with a list price of 4.25 million dollars, making it among the most expensive medicines ever approved. Onasemnogene abeparvovec, sold as Zolgensma, is a single infusion for spinal muscular atrophy priced near 2.1 million dollars. Exagamglogene autotemcel, known as Casgevy, the first CRISPR-edited cell therapy approved for sickle cell disease, and lovotibeglogene autotemcel, or Lyfgenia, a lentiviral-edited counterpart, list in a similar range. These figures dwarf the cost of conventional orphan drugs and raise immediate questions about affordability for health plans serving children.

Yet the authors caution against reading these prices as simple profiteering. The costs reflect the genuine expense of manufacturing an individualized cell or gene product, a process that involves harvesting a patient’s own cells, engineering them in specialized facilities, performing quality control, and returning them to the patient, all under regulatory scrutiny. Independent health economic assessments have judged several of these therapies cost-effective when measured against the lifetime value of a one-time cure compared with years or decades of standard care, which for conditions like sickle cell disease includes repeated hospitalizations, transfusions, and pain crises. From that perspective, a multimillion-dollar price tag can represent a bargain over a lifetime, even as it creates an acute budget shock for any single insurer.

This tension between long-term value and short-term affordability lies at the heart of the coverage problem. A child treated at age three may remain enrolled with a health plan for only a few years before the family changes jobs or coverage, meaning the insurer that pays for the cure may never capture the savings from avoided care. Economists describe this as a mismatch between who pays and who benefits, and it gives payers a rational incentive to delay or deny coverage in the hope that someone else will bear the cost. For families, the consequence is that access can depend on the accident of which insurance plan covers a child in a given year.

The commentary also examines the frontier of ultra-rare disease, where the economics become even more difficult. Landmark cases such as patient-specific in vivo gene editing for a rare genetic condition and patient-customized oligonucleotide therapy, each developed for a single individual, have demonstrated that bespoke treatments are technically feasible. But therapies designed for one or a handful of patients will never recoup their development costs through sales, no matter how high the price. The authors point to proposed funding approaches for N-of-1 trials of individualized gene-targeted therapies and to efforts to overcome barriers to commercially pre-viable gene and cell therapies for rare and ultra-rare diseases as signs that researchers and policymakers are searching for models that do not depend on conventional market returns.

Regulatory innovation is moving in parallel. The FDA has launched a framework for accelerating the development of individualized therapies for ultra-rare diseases, an acknowledgment that the agency’s traditional pathways were designed for drugs manufactured at scale rather than treatments built for one patient. On the payment side, the Centers for Medicare and Medicaid Services have expanded access to gene therapies through innovative state agreements, and proposals for a national benefit for cell and gene therapies have been advanced in health policy forums. These arrangements, including outcome-based contracts in which manufacturers receive full payment only if a therapy works as promised, represent early attempts to spread risk between manufacturers, payers, and the public.

For the pediatric community, the stakes are unusually high because timing matters in a way it does not in adult medicine. Many of the conditions targeted by gene editing are progressive, and the therapies work best, or only, when administered before irreversible organ damage occurs. Newborn screening programs increasingly identify affected infants in the first days of life, creating a narrow window in which regulatory approval, insurance authorization, manufacturing capacity, and clinical delivery must all align. A delay of months in coverage negotiation can convert a curative intervention into a palliative one, which is why the authors frame payment policy as an integral component of the therapy itself rather than an administrative afterthought.

The commentary closes with a call to build the policy environment deliberately rather than reactively. The authors argue that without coordinated action on coverage, reimbursement, and financing, the remarkable science described by Pan and colleagues will reach only a fraction of the children who could benefit, and that uncertain payment prospects may deter investment in therapies for the rarest conditions of all. The tools of molecular biology have, in their telling, largely been proven; the rulebook that determines who can use them is still being written, and the next chapter will be drafted not by geneticists but by policymakers, insurers, and the families advocating for their children.

Subject of Research: Insurance coverage and reimbursement policy for pediatric gene and cell therapies

Article Title: Editing the rulebook: building the policy environment for children to access gene therapy

Article References: Pulcini, C. D., Chua, K.-P., Conti, R. M., On behalf of the Pediatric Policy Council, Keller, D., Montez, K., Ragavan, M., Lorch, S., Plax, K., Cheng, T., Geme, J. S., Lakshmanan, A., & Mariani, T. J. (2026). Editing the rulebook: building the policy environment for children to access gene therapy. Pediatric Research. https://doi.org/10.1038/s41390-026-05588-y

Image Credits: AI Generated

DOI: 10.1038/s41390-026-05588-y

Keywords: gene therapy, CRISPR, pediatrics, health policy, insurance coverage, drug pricing, rare disease, sickle cell disease, spinal muscular atrophy, reimbursement, base editing, ultra-rare disease

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