CRISPR/Cas9: implication for modeling and therapy of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that destroys motor neurons in the spinal cord, brainstem, and motor cortex, typically leading within 2–5 years from symptom onset to weakness, atrophy, paralysis, respiratory failure, and death. It affects about 2–3 per 100,000 individuals globally, and prevalence has risen with improved diagnosis. Current treatments, such as riluzole, only modestly extend survival, underscoring the need for effective therapies. While most ALS is sporadic, 10–15% is familial, implicating genetics. Over 50 genes are associated with ALS, with SOD1, C9orf72, TARDBP (TDP-43), and FUS accounting for roughly 75% of familial cases. Elucidation of molecular pathways linked to these genes has revealed therapeutic targets and motivated development of ALS models and gene therapy approaches. Gene editing offers potential for correcting pathogenic mutations; among platforms (ZFNs, TALENs, CRISPR/Cas), CRISPR/Cas9 is particularly attractive due to ease and cost, and is being explored for ALS modeling and therapy. This review summarizes the evolution of gene editing tools, recent ALS models, and applications of CRISPR/Cas9 to ALS.

- Gene editing evolution: ZFNs and TALENs laid groundwork but are limited by design complexity, off-target effects (ZFNs), and delivery size (TALENs). CRISPR/Cas9 enables RNA-guided targeting, efficient multiplex editing, and broad adoption, though off-target activity and DSB-driven risks remain. Base editors (ABE/CBE) and prime editors offer DSB-independent edits with distinct limitations. - ALS model construction with CRISPR/Cas9: - Cell models: CRISPR-engineered ALS-relevant mutations or knockouts in HT22, iPSCs, BV2, Neuro2a, NSC-34, HeLa, and hESC lines, including UBQLN2 P497H, OPTN KO, FUS-Q290X, and AEG-1 deficiency, enabling mechanistic studies (e.g., TDP-43 mislocalization; UBQLN2-associated pathways). - Small animals: Zebrafish, Drosophila, C. elegans, and rodents edited to model ALS genes (e.g., VCP KO zebrafish; FUS knock-in rats R521C; C. elegans SOD1 and FUS knock-ins; TDP-43 conditional loss-of-function) reveal roles in DNA damage response, autophagy, motor neuron development, and neuromuscular function. - Large animals: CRISPR/Cas9 has revealed neurodegenerative phenotypes in other disease contexts (pigs/monkeys) more faithfully than rodents; no CRISPR ALS large-animal models reported yet, highlighting a need. - Therapeutic CRISPR/Cas9 applications in ALS: - SOD1: - In SOD1-G93A mice, CRISPR-mediated disruption of mutant SOD1 via AAV delivery reduced spinal cord SOD1, lessened muscle atrophy, delayed onset, improved motor function, and extended survival by about 28–30 days (Gaj et al., 2017). Other studies reported prolonged survival, reduced SOD1 inclusions by up to 40%, and improved neuromuscular function with base editing or novel AAV variants. - Patient iPSC models corrected by CRISPR restored phenotypes, aiding biomarker and pathway discovery. - Limitation: many interventions applied pre-symptomatically; efficacy in symptomatic models remains uncertain. - C9orf72: - Pathogenic G4C2 hexanucleotide repeat expansions cause gain-/loss-of-function toxicity. CRISPR/Cas9 excision of the repeat in patient iPSCs and in vivo rescued key disease mechanisms; AAV9 delivery of Cas9/gRNAs in C9orf72 models rescued partial ALS phenotypes. - dCas9 targeting of repeat DNA/RNA reduced repeat transcription and myotonia in models; dual-gRNA approaches showed limited off-targets while correcting expansions in vivo. - FUS: - CRISPR correction of patient iPSC FUS mutations (e.g., G1566A, H517Q) normalized cellular phenotypes and implicated MAPK (ERK/p38) activation as a key pathway; HDAC6 inhibition reversed axonal transport defects in FUS-ALS motor neurons. - TARDBP (TDP-43): - TDP-43 loss or mutation disrupts RNA metabolism, contributes to cytoplasmic toxicity, and impairs BDNF secretion and mitochondrial Ca2+ handling. CRISPR correction of M337V in patient iPSCs restored neuronal phenotypes, supporting TARDBP as a therapeutic target. - Clinical landscape: - Despite four approved therapies (riluzole, edaravone, PB-TURSO, dextromethorphan-quinidine), disease-modifying efficacy remains limited. Of 969 ALS trials (as of June 20, 2023), most have not yielded strong positive outcomes due to delivery challenges, CNS bioavailability, limited disease models, late diagnosis, biomarker gaps, and incomplete mechanistic understanding. - Safety/technology considerations: CRISPR/Cas9 off-targets, DSB-induced large deletions/rearrangements, p53 activation, and cytotoxicity are concerns; base editors avoid DSBs but can cause bystander edits and have edit-scope constraints; delivery limits (AAV packaging) and HDR efficiency remain challenges.

The review synthesizes how CRISPR/Cas9 advances address two central needs in ALS research: faithful disease modeling and gene-targeted therapeutics. Genome editing enables isogenic patient iPSC models and diverse animal models to dissect mechanisms of key ALS genes (SOD1, C9orf72, FUS, TARDBP), revealing roles in autophagy, DNA damage repair, RNA processing, and excitotoxicity. Therapeutically, in vivo editing of SOD1 and excision of C9orf72 repeat expansions ameliorate motor deficits and extend survival in mouse models, demonstrating target engagement and functional rescue. These findings validate gene editing as a platform for mechanistic discovery and as a potential one-time intervention to correct causal mutations and normalize downstream pathways. However, translation requires mitigating safety risks of DSB-based editing, improving delivery to the CNS, expanding editing modalities (base/prime editing), and testing efficacy in symptomatic stages and in larger, more clinically relevant animals. The review situates CRISPR within a broader therapeutic landscape that still lacks disease-modifying treatments, highlighting gene editing’s promise alongside the need for improved biomarkers, earlier diagnosis, and robust preclinical models.

This review collates recent progress using gene editing—especially CRISPR/Cas9—to model ALS and to target key genetic drivers (SOD1, C9orf72, TARDBP, FUS). In vitro and in vivo studies show that correcting or disrupting pathogenic mutations can reverse cellular and behavioral phenotypes, extend survival in mouse models, and elucidate disease mechanisms. Despite this promise, significant hurdles remain: off-target effects and genotoxicity, limited cargo capacity of delivery vectors, suboptimal HDR efficiency, and uncertainties about efficacy in symptomatic disease and across diverse genetic backgrounds. Future work should focus on: minimizing off-target and DSB-associated risks (e.g., high-fidelity nucleases, base/prime editing), optimizing CNS delivery (AAV engineering, LNPs, nonviral approaches), developing large-animal ALS models, expanding studies to later disease stages, and integrating robust biomarkers and early diagnostic tools to streamline clinical translation.

- Technological: CRISPR/Cas9 off-target effects; DSB-associated large deletions, chromosomal rearrangements, and p53 activation; cytotoxicity; limited AAV cargo capacity; low efficiency of HDR-mediated correction; base editors’ bystander editing and limited edit scope. - Model-related: Many therapeutic studies treat young/presymptomatic mice, leaving efficacy in symptomatic models uncertain; lack of large-animal ALS models limits translational relevance; some rodent models fail to show overt neurodegeneration. - Translational: Challenges in CNS delivery and bioavailability; potential immunogenicity; need for long-term safety data; heterogeneity of ALS genetics and phenotypes; limited biomarkers and late diagnosis complicate trial design and readouts.