---
title: "CRISPR correction of AS-NMD in splicing-factor mutant cancer: article unavailable — synthesis and"
id: "frontiers-in-immunology-12-crispr-mediated-correction-of-oncogenic-as-nmd-in-splicing-factor-mutant-cancer"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-12-crispr-mediated-correction-of-oncogenic-as-nmd-in-splicing-factor-mutant-cancer"
content_type: "clinical_feed_article"
specialty: "Infectious Disease"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1958532"
published_at: "2026-10-23T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# CRISPR correction of AS-NMD in splicing-factor mutant cancer: article unavailable — synthesis and
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-12-crispr-mediated-correction-of-oncogenic-as-nmd-in-splicing-factor-mutant-cancer
- **Specialty:** [Infectious Disease](https://medichelpline.com/clinical-feed/infectious-disease.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1958532)
- **Published At:** 2026-10-23T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The source article title centers on **CRISPR**-mediated correction of aberrant alternative splicing–linked nonsense-mediated decay (abbreviated **AS-NMD**) in cancers driven by mutant splicing factors. - The publicly available page content provided to this task did not include the manuscript text, so no experimental results, datasets, or numeric outcomes were available from the source. - The source did not report study design details in the supplied content: there were no explicit methods, dosing or editing strategies, target genes, cell models, animal data, or clinical endpoints included. - Mechanistic context is relevant: mutant splicing factors can generate aberrant transcripts that trigger **NMD**, potentially reducing expression of key tumor suppressors or altering antigenic peptide generation. - Gene-editing approaches such as **CRISPR** can in principle be applied to restore canonical splicing, remove cryptic splice sites, or correct driver mutations in splicing regulators — but the source did not specify which approach the authors used. - Safety considerations for genome editing in cancer include off-target edits, delivery vehicle toxicity, immune responses, and impacts on tumor heterogeneity; the source did not provide safety data. - Translational questions remain: how to deliver editing reagents to solid tumors, whether corrected transcripts persist in diverse tumor clones, and what immunologic consequences editing might produce. The article text supplied did not answer these questions. - For readers seeking the primary data and conclusions, the source page accessible in this task did not include the full article; consult the Frontiers in Immunology article URL for the complete manuscript and datasets.
## Clinical Analysis & Structured Key Points
Frontiers | CRISPR-mediated correction of oncogenic AS-NMD in splicing factor mutant cancer ORIGINAL RESEARCH article Front. Immunol. , 23 October 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1958532 Published in Frontiers in Immunology Cancer Immunity and Immunotherapy 7 impact factor 11.3 citescore Part of a Research Topic Regulation of Gene Expression by RNA Processing Steps in Cancers Submission open 25k views 13 articles Editor & Reviewers Edited by N K Naoyuki Kataoka Reviewed by P R Praveen Rajendran C X Chengtao Xu Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Figure 5 View in article Figure 6 View in article Figure 7 View in article Figure 8 View in article Figure 9 View in article ORIGINAL RESEARCH article Front. Immunol. , 23 October 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1958532 CRISPR-mediated correction of oncogenic AS-NMD in splicing factor mutant cancer P N Preeti Nagar 1,2 † M R Md Rafikul Islam 1,2 † N A Nirjhor Anuvob Rahman 1 S A Shabiha Afroj Heeamoni 1 M M Md Mahbub Hasan 1 S H Saaimatul Huq 1 F A Fariha Alam Bushra 1 M H Mengye Hu 1 R A Rezwan Ali 3 M H Mokarram Hossain 2,3 +2 more M A Mohammad Alinoor Rahman 1,2 * 1. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States 2. Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR, United States 3. Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock, AR, United States See more Article metrics View details Abstract Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) evolved as a master regulator of gene expression. Dysregulated AS-NMD has been identified as the root of many human maladies, from developmental defects to deadly cancer. Poison exons (PEs) are highly conserved alternative exons that contain a premature termination codon and elicit AS-NMD when included in a transcript. Cancer cells often exploit the inclusion of PEs to downregulate tumor suppressors or the exclusion of PEs to upregulate oncoproteins. Therefore, PEs have drawn significant attention as a novel therapeutic avenue for cancer and other diseases. It was previously reported that an oncogenic mutation in the splicing factor SRSF2 promotes downregulation of the tumor suppressor EZH2 by including a PE, resulting in altered chromatin regulation, defective hematopoiesis, and cancer development. Here, we examine a therapeutic proof-of-concept for manipulating PE-mediated oncogenic AS-NMD using a CRISPR-based approach. Using paired guide RNA, we successfully deleted the PE of EZH2 from the genome of SRSF2-mutated leukemia. This editing resulted in EZH2 mRNAs without a PE, and restored EZH2 protein expression. This subsequently restored Polycomb Repressive Complex 2 (PRC2) association, and global H3K27 histone methylation. The proposed CRISPR strategy provides compelling evidence for correcting oncogenic PE inclusion, highlighting its potential for therapeutic application against PE in cancer and other human diseases. Introduction The human genome evolved as a highly coordinated blueprint that preserves precise instructions for the appropriate regulation of gene expression, enabling diverse biological functions and responses to internal and external challenges. Alternative splicing (AS) and nonsense-mediated mRNA decay (NMD) are two post-transcriptional RNA processing mechanisms. They play crucial roles in regulating gene expression ( 1 – 3 ). AS promotes proteome diversity using a limited number of genes. NMD evolved as a surveillance mechanism to eliminate faulty transcripts with a premature translation termination codon (PTC), such as those generated by nonsense or frameshift mutations. AS also often generates mRNAs with a PTC, which are degraded by NMD. This coordinated RNA processing mechanism is called AS coupled to NMD (AS-NMD). Studies in recent decades have highlighted AS-NMD as a powerful regulator of gene expression, playing a pivotal role during development, cell proliferation and differentiation, metabolism, immune surveillance, and many other biological processes ( 4 , 5 ). About 95% of human multiexon genes undergo AS, and ~33% of alternatively spliced transcripts are targets for NMD ( 6 – 8 ). Dysregulated AS-NMD has been identified as the root of many human maladies, including developmental defects, neurological diseases, metabolic disorders, immune dysfunction, genetic abnormalities, and deadly cancer ( 4 , 5 , 8 – 11 ). AS-NMD can be exploited positively or negatively to drive pathological consequences. For example, AS-NMD can compromise cellular integrity by degrading proteins essential for physiological functions, immune surveillance, and tumor suppression. In contrast, evading or suppressing AS-NMD can compromise gene expression fidelity and may allow the expression of defective or pathogenic proteins, such as oncoproteins. Poison exons (PEs) are a class of highly conserved cassette exons that comprise PTCs and therefore promote AS-NMD when included in transcripts ( 12 – 14 ). Although poison exons are not used for protein-coding in host genes, they play an important role in controlling gene expression ( 5 , 14 , 15 ). For example, the serine-arginine-rich (SR) splicing factors (SFs) contain ultra-conserved PEs in their genes (SRSF1-SRSF12) ( 12 – 14 ). Although SR proteins are important for normal splicing regulation, their altered expression can turn them into oncoproteins ( 16 – 19 ). In normal cells, when SR protein levels are upregulated, SR proteins autoregulate their expression through AS-NMD by including PEs ( 12 – 14 ), thereby avoiding an effect in oncogenic transformation. Tumor cells often exploit PEs to differentially regulate AS-NMD in their favor. SR proteins are frequently overexpressed in various solid tumors ( 4 , 16 , 20 – 22 ), including breast, colon, lung, ovarian, liver, and pancreatic tumors. Recent studies have shown that upregulation of SR proteins in solid tumors is often correlated with reduced inclusion of PEs ( 14 , 15 ). In contrast to splicing factor overexpression, recurrent mutations in splicing factors (most frequent in SRSF2, SF3B1, U2AF1, and ZRSR2) are frequently identified in hematologic malignancies, such as myelodysplastic syndromes and leukemia, and are also common to different solid tumors ( 4 , 23 , 24 ). It has been reported that several important genes linked to hematopoiesis are downregulated by the inclusion of PEs ( 4 , 25 – 30 ). Therefore, PEs have received significant attention in recent years as potential therapeutic targets due to their strong links with various cancers and numerous human diseases. Strategies to manipulate PE-mediated AS-NMD are emerging in genetic diseases and cancer ( 4 , 5 , 31 , 32 ). However, therapeutic efficacy is often compromised by technological limitations. Antisense oligonucleotide (ASO) pharmacology is a popular strategy to modulate AS or AS-NMD ( 32 , 33 ). However, the efficacy of ASO is often compromised by delivery challenges, suboptimal dosing, stability issues, or low throughput. RNA-mediated interference (such as siRNA or shRNA) can’t change alternative splicing in a gene-specific manner. Small molecules often appear toxic due to off-target effects and fail in clinical trials ( 31 , 34 , 35 ). These disparities remain as critical roadblocks to therapeutic success. Therefore, research should focus on developing novel, effective targeted therapeutic approaches grounded in a comprehensive understanding of molecular regulations. Here, we examine a gene-specific targeted approach to modulate PE-mediated oncogenic AS-NMD using CRISPR/Cas9. Although CRISPR/Cas9 is frequently used to knock out a target gene, our strategy restores its expression by preventing the inclusion of a PE, thereby evading AS-NMD. As a proof-of-concept, we investigated a blood cancer model harboring an oncogenic mutation in the splicing factor SRSF2. We and others previously showed that mutations in SRSF2 (SRSF2 Mut ) promote aberrant inclusion of a PE in the tumor suppressor gene EZH2 and elicit AS-NMD ( 25 – 27 ). This results in downregulated EZH2 protein levels, altered chromatin regulation, defective hematopoiesis, and leads to the development of cancer ( 25 ). To challenge these molecular maladies, we exploited the CRISPR/Cas9 system with paired guide RNA (pgRNA) targeting the flanking intronic sequences of the PE of EZH2 in SRSF2 Mut model cancer cell lines. Genomic deletion of the poison exon corrected the EZH2 splicing error, generating a canonical transcript isoform that is not degraded by AS-NMD and restoring protein expression. This subsequently enhanced global histone H3K27 methylation. This proof-of-concept shows promising therapeutic potential targeting PE-mediated AS-NMD in cancer and many other human diseases. Materials and methods Cell culture SET2, MOLM-13, TF1, K052, K562, and K562 P95H(Mut) cells were cultured in RPMI-1640 medium with 1% penicillin streptomycin (Sigma) and supplemented with 10% fetal bovine serum (FBS) and maintained in an incubator at 37˚C with 5% CO 2 . HEK 293T cells were cultured in DMEM medium with 1% penicillin streptomycin (Sigma) and supplemented with 10% fetal bovine serum (FBS) and maintained in an incubator at 37˚C with 5% CO 2 . The sources of different cell lines are described in Supplementary Table S1 . Splicing analysis using reverse transcription PCR Total RNA was isolated using TRIzol reagent, followed by DNase treatment. Reverse transcription reaction was conducted using ImProm-II reverse transcriptase (Promega). During cDNA synthesis, an oligo (dT) reverse primer was used. PCR reaction was performed by Phusion High-Fidelity PCR Master Mix with HF Buffer (M0531L, New England Biolabs). The primer sequences are shown in Supplementary Table S2 . PCR products were separated on a 2% agarose gel and stained with SYBR Safe (Invitrogen). The gel image was captured using the ChemiDoc MP Imaging System (Bio-Rad). PCR bands were quantified, and the percent spliced-in (PSI) ratio was calculated as the exon-included isoform band intensity divided by the intensity of included and skipped isoform bands. To determine the linear range of RT-PCR, amplification conditions were set up using serial dilutions of cDNA template (20–200 ng) across a range of cycle numbers (18 to 30). We performed EZH2 RT-PCR using 100 ng cDNA template for 25 cycles. Protein expression analysis by western blotting For protein isolation, cells were washed and harvested in 1x PBS containing Protease Inhibitor Cocktail. After centrifugation at 2,000 x g for 5 min, the cell pellets were resuspended in buffer A [10 mM HEPES-NaOH (pH 7.8), 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 0.1% Nonidet P-40, Protease Inhibitor Cocktail], followed by incubation on ice for 30 min. The samples were then sonicated and centrifuged at 20,000 x g for 5 min. The supernatants were collected as total cell lysate for western blotting. The list of antibodies used is included in Supplementary Table S3 . Designing of pgRNA and cloning pgRNAs located in or near the targeted exon were designed according to the concept described previously ( 36 ), using both NAG and NGG protospacer-adjacent motifs. Candidate pgRNAs were cloned into the Lenti-multi-CRISPR vector (Addgene#85402) following a published method ( 37 ). All plasmids were sequenced using Sanger sequencing to confirm the desired sequences. pgRNAs used are listed in Supplementary Table S4 . Lentiviral transduction HEK 293T cells were cultured in DMEM medium with 10% FBS with antibiotics. The next day, the medium was replaced with fresh DMEM containing 10% FBS, chloroquine diphosphate, and no antibiotics. Cells were transfected with the Lenti-multi-CRISPR-pgRNA vector, pMD2.G, and psPAX2, and incubated at 37˚C for 6 h. The medium was replaced with fresh DMEM supplemented with 10% FBS and without antibiotics. After 48 h, the lentiviral supernatant was collected and filtered through 0.45 μm filters. K052 or K562 Mut cells were grown in RPMI-1640 medium with 10% FBS. The next day, K052 or K562 Mut cells were transduced with lentiviral supernatant containing polybrene, and the procedure was repeated with fresh virus the following day. The next day, cells were cultured in fresh medium supplemented with 1 μg/mL puromycin for 8 days, with the medium replaced every 2 days. Cells were then split into 96-well plates for single-clone expansion. Positive single clones were selected and confirmed following PCR and Sanger DNA sequencing of the genomic DNA. All experiments were performed with at least three independently generated CRISPR clones. EZH2 inhibitor EPZ-6438 (Tazemetostat) was used as an EZH2 inhibitor (EZH2i), purchased from SelleckChem (Cat# S7128). It was used according to the manufacturer’s instructions. Co-immunoprecipitations Control (empty vector) or pgRNA-edited K052 cells were grown in two 10 cm flasks. Co-IPs were performed on nuclear protein lysate as described previously with minor modifications ( 38 ). The SUZ12 antibody was added to the nuclear protein lysate, with gentle agitation at 4 °C for 2 h. Protein G Magnetic Beads (Sigma-Aldrich) were prepared according to the manufacturer’s instructions and incubated with the lysate with gentle agitation at 4 °C overnight. The IP samples were washed three times with IP buffer. Proteins for western blotting were eluted by boiling at 95˚C for 5 min. Real-time cell growth analysis We seeded control (empty vector) or pgRNA-edited K052 cells in standard 96-well tissue culture plates at densities of 625, 1,250, 2,500, or 5,000 cells per well in 200 μL of complete culture medium. We monitored cell growth using the xCELLigence RTCA eSight Live-Cell Analysis System (Agilent Technologies, Santa Clara, CA, USA), acquiring bright-field images at regular intervals for up to 480 h while maintaining cells at 37 °C in a humidified atmosphere with 5% CO 2 . We quantified bright-field confluency as a percentage using RTCA eSight image analysis software. Flow cytometry analysis Control (empty vector) or pgRNA-edited K052 cells were stained with propidium iodide (PI) (Cat# V13242, ThermoFisher Scientific) for cell cycle analysis and with Annexin V and PI for apoptosis analysis and evaluated using BD LSRFortessa-Flow Analyses according to the manufacturer’s instructions. We analyzed the data in FlowJo and compared the results with the control group. Antisense oligonucleotides-mediated splicing correction We synthesized ASO from Integrated DNA Technologies (IDT). The ASO2 sequences: 5’-TCCAACAGGCAATAT-3’ ( 39 ). We transfected K052 cells with ASO2 using Lipofectamine (Invitrogen). RNA or proteins were isolated 48 h later after the transfection. Quantification and statistical analysis Densitometric quantification of RT-PCR or western blotting was performed using ImageJ. Statistical analysis was performed using t-test or ANOVA, with post-hoc test where appropriate. Data are presented as mean and standard deviation and indicated by error bars. Graphs were generated using GraphPad Prism software. In statistical analyses, n=3 represents biological replicates using independently generated CRISPR clones. Results AS-NMD is a crucial mediator of tumorigenesis in splicing-factor-mutated cancers Heterozygous mutations in splicing factors have been recurrently identified in cancer patients with myelodysplastic syndromes (MDS), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and a variety of solid tumors ( 23 ). The most frequent mutations occur in SF3B1, SRSF2, U2AF1, or ZRSR2. These mutations typically alter RNA-binding properties, leading to genome-wide splicing alterations ( 25 – 30 , 40 – 47 ). Interestingly, certain mRNAs promoted by different mutant splicing factors comprise PTCs and are degraded by AS-NMD. Some of these events are linked with tumorigenesis and are critical vulnerable targets for cancer therapy ( 4 , 19 , 21 , 48 ). We previously analyzed the Cancer Genome Atlas AML patient RNA-sequencing data set (TCGA-LAML) with or without SRSF2 mutation (SRSF2 Mut vs. SRSF2 WT ) ( 26 ). We reanalyzed the data to include a potential oncogenic AS-NMD target for our investigation. Among 843 differential splicing events (DSE), the majority of the events were alternative cassette exon splicing (CE), and the others included alternative splice site selection (ASS) and alternative intron retention (IR) ( Supplementary Figure S1 ). Among these DSE, ~16% were AS-NMD targets. Again, the majority of the AS-NMD events represent CE ( Figures 1A, B ). Experimental validation of tumorigenic activity identified only a handful of targets, considered as cancer driver events, while others are passenger splicing events with no functional relevance to cancer development ( 25 – 27 , 49 ). Several consistent, noteworthy, and validated cancer driver events include: a poison-exon inclusion-mediated AS-NMD in EZH2; an intron-retention-mediated AS-NMD in INTS3; and an alternative-exon-exclusion-mediated AS-NMD in CLK3 ( 25 – 27 , 49 ). EZH2 (Enhancer of Zeste 2 Polycomb Repressive Complex 2 Subunit) is an enzyme that catalyzes methylation of histone H3 at lysine 27 (H3K27) and plays an important role in chromatin regulation ( 50 ), suppression of gene expression, stem cell differentiation and development, and determination of cell fate ( 50 ) ( Figure 1C ). Downregulation of EZH2 protein expression through AS-NMD, promoted by SRSF2 Mut , drives defective hematopoietic differentiation progressing to cancer ( 25 – 27 ). INTS3 is a member of the integrator complex 3, which functions in snRNA processing and RNA polymerase pause release. Downregulation of INTS3 protein expression through AS-NMD, driven by SRSF2 Mut , promotes increased stalling of RNA polymerase II (RNAPII) ( 27 ). CLK3 (CDC-like kinase 3) is a protein kinase that regulates pre-mRNA splicing by phosphorylating SR splicing factors. Aberrant skipping of an exon in CLK3 in SRSF2 Mut cancer generates splicing-derived neoantigens ( 49 ). Since the focus of our proof-of-concept is modulating the splicing of oncogenic poison/cassette exon using CRISPR/Cas9, we chose EZH2 in SRSF2 Mut blood cancer as our model oncogenic target. Figure 1 Genome-wide altered splicing profile promoted by oncogenic Pro95 mutations in SRSF2. (A) Proportion of differential AS-NMD events (promoted and repressed) analyzed from TCGA-LAML RNA-seq of AML patients (SRSF2 Mut vs. SRSF2 WT ) ( 26 ). ASS: alternative splice site selection; CE: cassette exon splicing; IR: intron retention. (B) Ranking of the top ten AS-NMD targets in promoted and repressed CE events, respectively. ΔPSI: differences in percent spliced-in (SRSF2 Mut vs. SRSF2 WT ). (C) Cartoon showing the function of EZH2 in histone methylation and chromatin regulation. PRC2: Polycomb Repressive Complex 2. (D) Left, diagram showing EZH2 poison exon (PE) splicing. Right, quantification of the EZH2 PE inclusion as percent spliced-in (PSI) in TCGA-LAML samples from the genotypes indicated on the right (mean ± SD, n=5 independent primary AML patient samples, ***p<0.001, unpaired two-sided t-test). (E) Left, representative RT-PCR gel showing the EZH2 PE splicing in different human leukemia cell lines from the indicated genotypes. Right, quantification of the EZH2 poison exon inclusion as PSI. Bar graph (mean ± SD, n=3 independent biological replicates). (F) Left, representative western blotting showing indicated protein expression in different human leukemia cell lines from the indicated genotypes. Right, quantification of the relative protein expression (RPE) of EZH2 normalized against GAPDH (EZH2/GAPDH). Bar graph (mean ± SD, n=3 independent biological replicates). Recapitulation of ab
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