The CRISPR/Cas9 system, derived from the adaptive immune system of bacteria, is a powerful genome-editing tool composed of two key components: The Cas9 endonuclease and a guide RNA (gRNA). The gRNA, comprising a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), directs Cas9 to a specific genomic sequence through complementary base pairing (6–8). Upon recognition of the target DNA adjacent to a protospacer adjacent motif (PAM), the Cas9 protein induces a double-strand break (DSB). Cells repair these DSBs through one of two major pathways: non-homologous end joining, which is error-prone and may result in insertions or deletions (indels), or homology-directed repair, which uses a homologous template to accurately repair the break. This dual repair mechanism allows researchers to either disrupt gene function or introduce precise genetic modifications (Fig. 2). The programmability, efficiency and simplicity of CRISPR/Cas9 have made it a cornerstone in both basic research and therapeutic development. Its ability to target virtually any locus in the genome with high precision marks a significant advancement over previous editing technologies (9–11).

CRISPR/Cas9 has transformed cancer research by enabling the functional interrogation of oncogenes and tumor suppressor genes, modeling of tumor evolution and developing treatment strategies (Fig. 3) (12–14). In NSCLC, it has facilitated the creation of genetically engineered cell lines and animal models that mirror specific mutations found in patients, such as EGFR, KRAS, or ALK alterations. These models are critical for studying tumorigenesis, drug response and resistance mechanisms. Moreover, CRISPR/Cas9 is widely used in high-throughput genetic screening to identify genes that mediate resistance or sensitivity to targeted therapies (15–17). These screens can uncover novel synthetic lethal interactions and inform rational combination therapies. As a result, CRISPR-based approaches are becoming indispensable in efforts to overcome therapy resistance in NSCLC and other malignancies.

Compared with earlier gene-editing platforms such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 offers several advantages (18). It is easier to design, as it relies on RNA-based targeting rather than engineered protein-DNA interactions. This design flexibility accelerates experimentation and reduces cost (19). CRISPR/Cas9 also supports multiplex editing, targeting multiple genes simultaneously using different gRNAs, which is not easily achievable with ZFNs or TALENs (20). Despite its advantages, challenges remain, particularly with off-target effects, where the Cas9 nuclease may cut unintended genomic sites. However, the development of high-fidelity Cas9 variants and improved gRNA design algorithms have substantially mitigated these concerns (21–23). As the technology matures, CRISPR/Cas9 continues to expand its role from a basic research tool to a promising platform for clinical gene therapy, especially in oncology.

The KRAS gene, a member of the RAS family of small GTPases, plays a critical role in cell proliferation, differentiation and survival. It is the most prevalent oncogenic driver mutation in NSCLC, observed in 20–30% of cases, with nearly 97% of mutations occurring as point mutations in codon 12 or 13 of exon 2 (24). These mutations result in constitutive activation of KRAS, leading to persistent downstream signaling through pathways such as MAPK and PI3K/AKT, ultimately promoting tumorigenesis and metastasis (25,26). The high prevalence of KRAS mutations in NSCLC underscores its importance as a therapeutic target and highlights the need for innovative strategies to inhibit its aberrant activity. However, the targeting of KRAS mutations in the treatment of lung cancer poses a significant challenge primarily due to the intricate structure and functionality of the KRAS protein, rendering it resistant to drug intervention. According to KRYSTAL-1 trial, 45% of patients developed resistances to KRAS inhibitor (27,28). Consequently, lung cancer patients harboring KRAS mutations experience limited treatment efficacy and the emergence of resistance is a prevalent concern (29). Consequently, patients with KRAS-mutated NSCLC have traditionally had limited treatment options and poor outcomes. However, recent advances have led to the development of covalent inhibitors specifically targeting the KRAS G12C mutation, a subset found in NSCLC. Notably, sotorasib (30) and adagrasib (28), demonstrated promising clinical efficacy and have been approved for use in KRAS G12C-mutated NSCLC. In a pivotal study, researchers employed a genome-scale CRISPR interference platform to systematically identify genetic vulnerabilities in KRAS G12C-mutant lung and pancreatic cancer models treated with KRAS G12C inhibitors (15). The study revealed ‘collateral dependencies’; genes whose loss increased cellular sensitivity to KRAS inhibition. These findings enabled the identification of two classes of combination therapies that either enhanced target engagement or inhibited compensatory survival pathways, offering a framework for rational therapeutic combinations to overcome resistance. Further advancing CRISPR applications, Gao et al (31) developed two CRISPR systems, CRISPR/SpCas9-mediated genome editing and a transcriptional repression system using dCas9-KRAB, to selectively target the KRAS G12S mutant allele. The study demonstrated effective suppression of mutant KRAS expression in A549 cells, leading to reduced phosphorylation of downstream effectors Akt and ERK, impaired tumor cell proliferation and significant tumor regression in vivo. Notably, the aforementioned analysis suggested that this mutation-specific editing strategy could be extended to a broader range of oncogenic mutations. Additionally, Dompe et al (32) used CRISPR screening to identify MAPK7 as a key therapeutic target in KRAS-mutant tumors. The authors' findings showed that combined inhibition of MAPK7 and MEK resulted in synergistic suppression of tumor growth in KRAS-mutant NSCLC xenograft models, highlighting the therapeutic potential of co-targeting downstream signaling nodes to overcome resistance. Together, these studies demonstrate the transformative potential of CRISPR/Cas9 technology in dissecting the molecular underpinnings of KRAS-driven NSCLC and in informing the development of novel targeted therapeutic strategies.

The epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase that plays a central role in regulating cell proliferation, survival and differentiation. Activating mutations in EGFR are among the most common driver alterations in NSCLC, occurring in 10–20% of patients. These mutations, particularly exon 19 deletions and the L858R point mutation in exon 21, are associated with sensitivity to EGFR tyrosine kinase inhibitors (TKIs) (15,16,33). The introduction of first-generation EGFR-TKIs, such as gefitinib (34) and erlotinib (35), marked a paradigm shift in the treatment of advanced NSCLC. Subsequent generations of TKIs, including icotinib (36), afatinib (37), dacomitinib (38), osimertinib (39), almonertinib (40) and furmonertinib (41), further improved patient outcomes (Table I). Although EGFR-TKIs provide significant initial clinical benefits for patients with EGFR-mutated lung cancer, 50–60% of these patients eventually develop acquired resistance to first- or second-generation EGFR-TKIs. Moreover, 20–30% of EGFR-mutated patients exhibit primary resistance when treated with third-generation EGFR-TKIs as a first-line therapy (42–46). Common resistance mechanisms include secondary mutations in EGFR (such as T790M and C797S), activation of bypass pathways (such as MET amplification) and histological transformation to small cell lung cancer (47–50). The advent of CRISPR/Cas9 technology has provided a powerful platform to investigate these resistance mechanisms and develop strategies to overcome them. For example, Park et al (51) employed CRISPR/Cas9 to introduce the mTOR L1433S mutation into NSCLC cells, revealing that activation of the AKT signaling pathway may mediate resistance to third-generation EGFR-TKIs, such as osimertinib. The findings further demonstrated that dual inhibition of EGFR and mTOR signaling could restore drug sensitivity. Park et al (51) underscored the importance of CRISPR/Cas9 technology in uncovering innovative resistance mechanisms to EGFR TKIs. In another study, CRISPR/Cas9-mediated knockout of ZEB1 and FGFR1 identified their crucial roles in epithelial-mesenchymal transition (EMT)-associated resistance to EGFR-TKIs (52). Inhibition of ZEB1 impaired EMT processes and resensitized NSCLC cells to EGFR inhibitors, while targeting FGFR1 suppressed survival pathways activated during resistance development. Zeng et al (53) used genome-wide CRISPR/Cas9 screening to elucidate the role of RIC8A in modulating YAP pathway activity. Loss of RIC8A decreased the synergistic cytotoxic effects of EGFR-TKIs, highlighting potential targets for enhancing drug efficacy. Moreover, CRISPR/Cas9 was used to model the C797S mutation in EGFR, a key driver of resistance to third-generation TKIs. Functional studies revealed that overexpression of AXL contributed to reduced sensitivity to AZD9291 (osimertinib) and that AXL inhibition may represent a viable therapeutic strategy for C797S-mutant tumors (54). Finally, CRISPR/Cas9 barcoding technology has enabled tracking of heterogeneous subpopulations of NSCLC cells harboring specific resistance mutations (55). This approach allows dynamic modeling of resistance evolution under drug pressure and evaluation of combination therapies, offering important insights for optimizing treatment strategies. Collectively, these studies highlight the critical role of CRISPR/Cas9 in dissecting the complex molecular landscape of EGFR-TKI resistance in NSCLC and underscore its potential in identifying novel therapeutic vulnerabilities.

The ALK gene, located on chromosome 2p23, encodes a receptor tyrosine kinase that is part of the insulin receptor superfamily. In NSCLC, ALK gene rearrangements are found in 3–7% of cases and are considered key oncogenic drivers (56). Among the identified ALK fusion partners, EML4-ALK is the most common variant, accounting for ~85% of ALK-rearranged NSCLC cases (57). The fusion leads to constitutive activation of the ALK kinase domain, promoting uncontrolled cell proliferation and survival through downstream signaling pathways such as MAPK/ERK and PI3K/AKT (58,59). Crizotinib, the first ALK inhibitor, was approved by the FDA in 2011 and markedly improved outcomes for ALK-positive NSCLC patients (60–63). However, resistance to crizotinib and subsequent generations of ALK inhibitors, including ceritinib (64), alectinib (65), brigatinib (66) and lorlatinib (67) (Table II), commonly develops, limiting long-term efficacy (68). Previous studies have shown that ~40% of ALK-mutant patients develop resistance to ALK-TKIs, with ~10% exhibiting primary resistance and 30% showing acquired resistance (69,70). Resistance mechanisms to ALK-TKIs are diverse and include secondary mutations within the ALK kinase domain, gene amplification and activation of alternative signaling pathways such as EGFR, KIT and IGF1R (71,72). To investigate these mechanisms, researchers have employed CRISPR/Cas9-based genome editing to create precise models of ALK gene rearrangements in vitro (55). In a landmark study, Maddalo et al (73) used CRISPR/Cas9 to generate a mouse model harboring the EML4-ALK fusion, effectively recapitulating the genetic, histological and molecular features of ALK-rearranged NSCLC. These genetically engineered mice developed tumors that responded to ALK inhibitors, providing a robust platform for evaluating drug efficacy and resistance mechanisms in vivo. These genetically engineered mice developed tumors that responded to ALK inhibitors, providing a robust platform for evaluating drug efficacy and resistance mechanisms in vivo.

The ROS1 gene, located on chromosome 6q22, encodes a receptor tyrosine kinase that belongs to the insulin receptor family. ROS1 rearrangements represent oncogenic fusion events in 1–2% of NSCLC cases and are considered actionable driver mutations (58–61). Due to structural homology between the kinase domains of ROS1 and ALK, several ALK inhibitors, including crizotinib, entrectinib, ceritinib and lorlatinib, have demonstrated efficacy against ROS1-rearranged NSCLC (74–81). While these targeted therapies provide significant initial clinical benefit, the emergence of acquired resistance remains a major barrier to long-term disease control. Resistance mechanisms include on-target mutations within the ROS1 kinase domain, such as G2032R and D2033N, that impair inhibitor binding, as well as less well-characterized off-target or bypass pathway activations (74,75). To improved understand the biology of ROS1 fusions and associated resistance mechanisms, researchers have employed CRISPR/Cas9 technology to model ROS1 rearrangements in vitro. Choi and Meyerson (82) designed single guide RNAs (sgRNAs) targeting intron 6 of CD74 and intron 33 of ROS1, successfully inducing a CD74-ROS1 translocation via CRISPR-mediated chromosomal inversion. This model provided a reliable platform for evaluating fusion-driven signaling and drug response. Sato et al (83) engineered EZR-ROS1 fusions in HBECp53 lung adenocarcinoma cells using CRISPR/Cas9. The resulting cells exhibited hyperactivation of the MEK/ERK signaling pathway, implicating this axis in both primary and acquired resistance to ROS1-targeted therapies. Treatment with a combination of the MEK inhibitor selumetinib and crizotinib markedly suppressed cell proliferation in vitro and in vivo, offering a promising therapeutic strategy.

The MET proto-oncogene, located on chromosome 7q31, encodes the MET receptor tyrosine kinase, a key component of the hepatocyte growth factor (HGF)/MET signaling axis. Upon HGF binding, MET dimerizes and undergoes autophosphorylation, activating downstream pathways such as PI3K/AKT, RAS/ERK and Wnt/β-catenin, which regulate cell proliferation, survival, motility and invasion (84–86). In NSCLC, aberrant MET signaling is observed in 5–20% of patients and may result from gene amplification, exon 14 skipping mutations, or protein overexpression (87). MET alterations serve as both primary oncogenic drivers and mechanisms of acquired resistance to EGFR-TKIs, making MET a critical therapeutic target. Several MET inhibitors, such as crizotinib, tepotinib, capmatinib and cabozantinib, have been approved or are under clinical investigation (88,89). Despite these advances, resistance to MET-targeted therapies remains a challenge. CRISPR/Cas9 technology has been key in modeling MET-driven oncogenesis and resistance mechanisms. For example, Togashi et al (90) used CRISPR/Cas9 to introduce MET exon 14 skipping mutations in 293 cells. The edited cells exhibited increased MET protein expression, enhanced phosphorylation and heightened sensitivity to crizotinib, supporting the oncogenic potential of MET exon 14 skipping (METex14) alterations and their role as therapeutic targets. In another study, Fernandes et al (91) employed CRISPR/Cas9 to generate isogenic models of METex14 in non-transformed human lung cells. These engineered cells displayed increased tumor sphere formation, motility and invasiveness in an HGF-dependent manner. When transplanted into NSG-hHGF knock-in mice, METex14 cells formed tumors, confirming their oncogenic capability in vivo. These models also proved valuable for evaluating the efficacy of MET inhibitors and understanding resistance mechanisms. Together, these findings highlight the utility of CRISPR/Cas9 in precisely modeling MET alterations in NSCLC. Such models enable mechanistic studies of MET-driven tumorigenesis and facilitate preclinical testing of targeted therapies, ultimately contributing to the development of more effective treatment strategies.

BRAF is a serine/threonine kinase and a key effector molecule of the MAPK/ERK signaling pathway. BRAF mutations occur in ~4% of NSCLC cases (92). Of BRAF mutations, ~50% are BRAF (V600E) (93). The BRAF (V600E) mutation leads to constitutive activation of the BRAF kinase, independent of upstream RAS signaling. This mutation enhances kinase activity by stabilizing the active conformation through a salt bridge with residue K507, resulting in sustained activation of downstream MEK-ERK signaling and promoting oncogenesis (94,95). Targeted therapies such as vemurafenib and dabrafenib have demonstrated clinical efficacy in BRAF-mutant cancers, including NSCLC. However, resistance to these inhibitors frequently emerges, driven by compensatory signaling or secondary molecular alterations (96,97). For example, reactivation of EGFR signaling or loss of the BRAF (V600E) allele has been associated with treatment failure, highlighting the need for a deeper understanding of resistance mechanisms (98). Recent CRISPR/Cas9-based studies have provided valuable insights into the biology of BRAF-mutant NSCLC. Vaishnavi et al (99) used CRISPR/Cas9 to knock out RBMS3, an RNA-binding protein, in BRAF (V600E)-driven lung-like organoids. Loss of RBMS3 enhanced tumorigenicity and resulted in micro-papillary histological features. Moreover, RBMS3 deletion conferred resistance to combination therapy with dabrafenib and trametinib, mediated through activation of the Wnt/β-catenin signaling pathway. These findings underscore the role of RBMS3 as a tumor suppressor and modulator of therapeutic response in BRAF-mutant NSCLC. CRISPR-based functional genomics thus enables precise dissection of resistance pathways and identification of novel co-targetable vulnerabilities. Collectively, CRISPR/Cas9 technology has expanded our understanding of BRAF-driven lung cancer by facilitating the development of robust experimental models and uncovering mechanisms of resistance that may guide the design of future combination therapies.