Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a progressive neurodegenerative disease that destroys motor neurons (MNs) in the spinal cord, brainstem, and motor cortex. ALS within 2–5 years of the onset of symptoms, leading to muscle weakening, atrophy, paralysis, respiratory failure, and death (Marcus, 2022). Additionally, ALS is one of the most common adult motor neuron diseases, affecting 2–3 persons out of every 100,000 individuals globally (Oskarsson et al., 2018; Feldman et al., 2022). Evidence shows that the prevalence of ALS has been rising in recent years, which may be largely attributed to the advancements in clinical services that allow precise diagnosis (Logroscino et al., 2018). However, ALS is still incurable. Current medications, such as Riluzole, prolong patient survival by an average of 3 months but are only modestly beneficial, highlighting the need for solutions to treat this dangerous condition.

Most cases of ALS are sporadic and have an unknown etiology, whereas approximately 10 ~ 15% of patients have a family history of the disease, strongly indicating a genetic predisposition (Sahdeo and Goldstein, 2021). The genetic etiology of ALS has been linked to more than 50 genes that include causal or disease-modifying variations (Taylor et al., 2016; Giovannelli et al., 2023). The most commonly investigated ALS genes, including superoxide dismutase (SOD1), chromosome 9 open reading frame 72 (C9orf72), TAR DNA-binding protein 43 (TDP43), and RNA binding protein fused in sarcoma (FUS), account for roughly 75% of familial ALS cases (Owens, 2017; Fang et al., 2022). Our understanding of the pathophysiology of ALS has significantly increased due to the analysis of the molecular pathways underlying these mutant ALS genes, revealing new information about prospective therapeutic targets. Accordingly, developing models of ALS and targeting the mutant genes (i.e., gene therapy) has been investigated worldwide to translate these gene therapies into the clinical setting.

With a corrected pathogenetic mutation, gene editing offers a potential treatment for ALS, and it is now beginning to be used in clinical settings (Mullard, 2019). In particular, the genome-editing technology known as clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9) has gained attention for its possible benefits in the management and treatment of ALS (Cho et al., 2013; Yamashita et al., 2013). Compared to other genome editing techniques like transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs), CRISPR/Cas9 is easy to use and reasonably priced (Yang et al., 2017; Bhardwaj et al., 2022; Meijboom et al., 2022). In this review, we emphasize the history of the emergence of gene editing tools and highlight the developments in CRISPR/Cas9 mediated gene correction and disease models gene therapy for ALS disease.

Gene editing constantly evolves to provide more precise, diverse, and large-scale implementations, allowing us to gain innovative insights into the biological field. Previously, homologous recombination (HR) and non-homologous end-joining (NHEJ) were used to carry out genetic engineering. Due to its low efficacy in many cell types and animals, we still need another alternative. Currently, there exist three main gene editing technologies: ZNFs, TALENs, and the CRISPR/Cas system, among which CRISPR/Cas system is now the most predominant technology.

Given that there are no ethical considerations, the experimental time is relatively short, and the expense is minimal, cell models are commonly used to explore many neurological illnesses, including ALS. Furthermore, recent advances in CRISPR/Cas9 technology allow researchers to precisely target and introduce modifications in the genomic DNA of different cell lines (Kramer et al., 2018; Karginov et al., 2023). The introduction of the CRISPR/Cas9 system has facilitated the development of numerous ALS cell models, such as HT22, iPSCs, BV2, Neuro 2a, NSC-34, hESC, and HeLa cell lines, etc. For example, Liu et al. (2022) recently used CRISPR/Cas9 gene-editing technology to create an astrocyte elevated gene-1 (AEG-1) deficient HT22 cell model that can be used to study AEG-1 related ALS. In addition, Zhang et al. (2022) used CRISPR/Cas9 to introduce a UBQLN2 P497H mutation into a healthy control iPSC line. Following this, the mutated iPSC line could differentiate into motor neurons, which can help comprehend the pathology of UBQLN2-related ALS (Zhang et al., 2022). Importantly, CRISPR-iPSC-based disease modeling could expedite research on ALS (Sen and Thummer, 2022) by targeting SOD1 (Kim et al., 2020), TDP-43 (Burkhardt et al., 2013), FUS (Wang et al., 2017), and C9ORF72 (Cali et al., 2019; Ababneh et al., 2020; Krishnan et al., 2020) genes, etc. Furthermore, Strohm et al. (2022) also made CRISPR/Cas9-engineered HeLa cells with ALS UBQLN2 mutations to determine how UBQLN2 mutations cause ALS. ALS can be caused by OPTN gene mutations, which result in cytoplasmic mislocalization, ubiquitination, and accumulation of nuclear TDP-43. Prtenjaca et al. (2022) used CRISPR/Cas9 methodology to create OPTN knockout (KO) cell lines for Neuro 2a and NSC-34 neurons and BV2 microglia to investigate the relationship between optineurin and TDP-43. Their research showed that OPTN mutations might lead to elevated levels of TDP-43 protein in microglia when optineurin is absent (Prtenjaca et al., 2022). In addition, Zhang et al. (2022) created a heterozygous FUS-Q290X knock in the human embryonic stem cell (hESC) line, which will be a valuable tool for investigating the disease mechanisms of ALS using the CRISPR/Cas9 system.

CRISPR/Cas9 technology is not only helpful in creating ALS cell models but also in creating ALS animal models. For instance, ALS can be caused by mutations in valosin-containing protein (VCP). On the other hand, there is no constitutive VCP knockout animal model due to embryonic lethality. Voisard et al. (2022) developed a constitutive CRISPR/Cas9-induced VCP knockout zebrafish model. Furthermore, Wall et al. (2021) created the Drosophila knockin models using the CRISPR/Cas9 technique to investigate VCP diseases (including multisystem proteinopathy and ALS) (Wall et al., 2021). To investigate the pathogenic effects of TP73 mutations in ALS, Russell et al. (2021) utilized CRISPR/Cas9 technology targeting TP73 in zebrafish and assessed motor neuron quantity and axonal structure. Their findings revealed that knocking out TP73 in zebrafish using CRISPR/Cas9 resulted in disrupted motor neuron development and abnormal axonal morphology, indicative of ALS pathology (Russell et al., 2021). Additionally, Bose et al. (2019) developed a TDP-43 loss-of-function model in zebrafish using the CRISPR/Cas9 system. Moreover, to examine mechanisms underlying mutations in FUS that lead to neuronal dysfunction in ALS patients, the C. elegans knockin models were generated by Baskoylu et al. (2022) using CRISPR-Cas9-mediated genome editing. Their findings suggested impaired autophagy might play a role in the neuromuscular defects and protein homeostasis associated with ALS FUS knockin animals (Baskoylu et al., 2022). As for the FUS gene, Zhang et al. (2018) developed a novel knockin rat model expressing a Fus point mutation (R521C) via CRISPR/Cas9. They discovered that FUS might regulate sleep, circadian rhythms, and learning and memory behavior (Zhang et al., 2018). Degeneration of neurons in many ALS subtypes has been linked to DNA damage and defective repair, but the underlying mechanisms are unclear. The role of TDP-43 in DNA damage response (DDR) has not been explored, despite growing evidence suggesting the involvement of other RNA/DNA binding proteins like FUS in DDR (Wang et al., 2018). Mitra et al. (2019) demonstrated that the loss of nuclear TDP-43 is linked to DNA double-strand break repair defects and DDR signaling in ALS by using CRISPR/Cas9-mediated conditional depletion of TDP-43 in C. elegans. Also, Baskoylu et al. (2018) made ALS knockin models in C. elegans by editing the endogenous Sod-1 gene with CRISPR/Cas9 and discovered that knockin models could replicate the neurotransmitter-type specificity of ALS. Using the CRISPR/Cas9 system, Kao et al. (2020) created MATR3 S85C knockin mice that mimic early-stage ALS and would be useful in future basic and preclinical research. Sporadic ALS patients were found to have mutations in the CREST gene (also called SS18L1), which acts as a calcium-regulated transcriptional activator. Loss of CREST causes neuroinflammatory responses and ALS-like motor defects in mice, as demonstrated by the work of Cheng et al. (2019), who used the CRISPR/Cas9 system to create CREST knockout and Q394X knockin mice. In addition, C9orf72-deficient mice (Sullivan et al., 2016) have been successfully established by using the CRISPR/Cas9 system due to the recent discovery of hexanucleotide repeat expansion in the C9orf72 gene as a major cause of frontotemporal lobar degeneration (FTLD) with ALS (DeJesus-Hernandez et al., 2011).

Researching the etiology and developing treatments for human diseases requires animal models. The discovery of specific gene mutations that cause neurodegenerative disorders has led to the development of numerous models in small animals with symptoms similar to those experienced by people with the diseases. Genetically modified rodents are today’s most widely used animal models because they apply to explore pathogenesis. However, most genetically modified rodent models do not exhibit overt neurodegeneration, creating difficulties in using them to rigorously test the effects of therapies on neurodegeneration (Philips and Rothstein, 2015). Recent research using CRISPR/Cas9-targeted large animals (such as pigs and monkeys) (Li et al., 2021; Yang et al., 2022) has uncovered important pathological events that resemble neurodegeneration in the patient’s brain. Still, these events could not be produced in small animal models. Injections of multiple sgRNAs are more effective than injections of a single type of sgRNA at a high concentration for achieving complete gene knockout. The complete gene knockout by the CRISPR/Cas9 system is conducive to reducing the test times of large animals and ensuring their accuracy, which is strongly supported by ethical considerations (Zuo et al., 2017). Although no ALS models in large animals have been developed so far using CRISPR/Cas9 technology, it is essential to create a large animal ALS model using this approach to gain more knowledge about the pathogenesis of ALS.

CRISPR/Cas9 system, used in cells and animal models, is a promising tool for modeling and treating genetic diseases (Table 2). Compared with cellular-level studies, animal models are more favorable for exploring the pathogenic gene and molecular pathways of diseases at the organismal level. For ALS, definite treatments are still lacking. Here, we mainly focus on CRISPR/Cas9 system applications in ALS. The great advantage of this strategy is that correction of the mutant DNA eliminates abnormal downstream pathways and is a one-time intervention theoretically. Currently, limited studies have been accomplished in the ALS field.

Although, four therapies (riluzole, edaravone, sodium phenylbutyrate and taurursodiol, and dextromethorphan-quinidine) have been approved now, but they could not stops or reverses disease progression (Meijboom and Brown, 2022). Using ALS as the search term, 969 clinical trials were gathered from clini-caltrials.gov on 20 June 2023. Unfortunately, the bulk of ALS clinical trials have failed to show any promising outcomes during the past two decades. This is partially caused by the therapeutic agents’ low bioavailability, ineffective therapeutic delivery to the CNS, difficulties with therapeutic administration, dearth of clinically relevant disease models, lack of late diagnosis, effective biomarkers, and inadequate understanding of the underlying molecular mechanisms causing the disease (Johnson et al., 2022). Therefore, new therapies and diagnostics are urgently needed to help with therapy and early diagnosis. All the current worldwide Phase III ALS clinical trials that are being summarized in Table 3.

We discussed numerous recent studies that have implicated gene editing, especially CRISPR/Cas9, to investigate the pathophysiology of ALS by animal models or iPSCs in this review. There remain limitations with these approaches, such as off-target effects, limited size of delivery vehicle, and low efficiency of gene correction through the HDR mechanism (Modell et al., 2022).

Over 50 genes, in which SOD1, C9orf72, TARDBP, and FUS are commonly genetic alterations, are listed as ALS-associated genes. Studies using CRISPR/Cas9-mediated in vivo genome editing of SOD1, C9orf72, TARDBP, and FUS functions show that this technique can identify the underlying molecular mechanism and reverse the pathologic phenotype in patient-derived disease models. These studies suggest that CRISPR/Cas9 system may play a potential role in gene therapy for ALS. However, more thorough studies are still needed to investigate the various underlying mechanisms of ALS to determine the treatment for ALS.

YS, YZ, and LL wrote the manuscript with support from QG, DY, and MS. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Key R&D Program of China (2022YFC2703700 and 2019YFA0802600) and the National Natural Science Foundation of China (81974244 and 81570960).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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