image: This figure illustrates the evolution of CRISPR technology from 1987 to 2019, presented in a horizontal timeline format and categorized into four generations, each denoted by a distinct color: The first generation (blue) represents DNA double-strand break editing technology, initiated with the establishment of the SpCas9 system in 2007 and subsequently refined for precision through advances such as the Cas9-D10A nickase and the high-fidelity SpCas9-HF1 in 2015; The second generation (orange) refers to targeted DNA cutting technology, including Cas12, developed in 2015, and Cas14, invented in 2018, which overcame PAM sequence restrictions, thereby enabling a broader target range and expanding applications from gene editing to molecular-level diagnostics; The third generation (green) involves targeted RNA regulation technology, encompassing the CRISPRi/a system for transcriptional regulation, the Cas13 system, invented in 2016, for the specific degradation and modification of cancer-related mRNA, and dCas9-DNMT3A/TET1-mediated DNA methylation editing developed between 2022 and 2023; The fourth generation (yellow) encompasses precise editing technologies that avoid double-strand breaks, including base editors (CBE/ABE), developed in 2016, and the Prime Editor, introduced in 2019, which have broadened applications to include the repair of diverse point mutations and the correction of tumor driver genes such as EGFR.
Credit: Kailai Li，Peixin Huang，Yue Qian，Anqi Lin，Jingjun He，Junyi Shen，Li Chen，Kai Miao，Jian Zhang
CRISPR gene-editing technology, renowned for its precision, is revolutionizing oncology research and treatment. Its evolution spans from the initial DNA double-strand break-inducing Cas9 to more advanced systems like Cas12, Cas13, base editors, and prime editors. These tools have expanded the scope of CRISPR beyond simple gene knockout to include RNA editing, transcriptional regulation, and epigenetic reprogramming, establishing a versatile platform for precision cancer interventions.
The generational progression of CRISPR systems has directly fueled innovations in cancer research. First-generation Cas9 enabled foundational gene knockout studies. Subsequent generations introduced Cas12 for DNA targeting with different PAM requirements, Cas13 for RNA degradation, and base/prime editors for precise nucleotide changes without inducing double-strand breaks. This functional expansion allows researchers to systematically investigate cancer mechanisms, from identifying driver genes to modeling drug resistance.
A primary application of CRISPR in oncology is the systematic identification of tumor driver genes and synthetic lethal targets through high-throughput screening. Genome-wide CRISPR libraries, such as GeCKO, have been instrumental in pinpointing genes essential for cancer progression, metastasis, and drug resistance across various cancer types. Furthermore, technologies like Perturb-seq, which combines CRISPR perturbation with single-cell RNA sequencing, enable the mapping of gene regulatory networks and heterogeneous cellular responses at single-cell resolution. CRISPR is also pivotal in dissecting the tumor microenvironment (TME) and immune evasion mechanisms. It is used to study metabolic reprogramming by targeting enzymes like LDHA, modulate angiogenesis via genes like VHL, and disrupt immune checkpoints such as PD-L1 and CD47. By editing these components, CRISPR helps reveal how tumors evade immune surveillance and suggests strategies for TME remodeling to enhance anti-tumor immunity.
In therapeutic development, CRISPR enables precise targeting of oncogenes and reactivation of tumor suppressor genes. Strategies include inactivating fusion oncogenes like BCR-ABL and EML4-ALK, or restoring the function of TP53 and PTEN. A major focus is engineering immune effector cells; CRISPR enhances CAR-T and NK cell therapies by knocking out inhibitory receptors (e.g., PD-1, TGFBR2) to improve persistence and cytotoxicity within the immunosuppressive TME, and facilitates the development of universal allogeneic cell products.
The effective delivery of CRISPR components remains a critical challenge. Viral vectors, such as AAV and lentivirus, offer high efficiency but face issues like immunogenicity and limited packaging capacity. Non-viral vectors, particularly lipid nanoparticles (LNPs), provide a safer alternative with lower immunogenicity and reduced risk of genomic integration, though their transfection efficiency needs improvement. Innovations in smart delivery systems, including microenvironment-responsive and spatiotemporally controlled nanocarriers, are being developed to enhance targeting specificity and safety.
In summary, the field is advancing through the development of next-generation tools like compact Cas enzymes (e.g., CasΦ, Cas12f) for easier delivery, and AI-guided sgRNA design platforms (e.g., DeepCRISPR) to optimize efficiency and minimize off-target effects. Clinical trials are already evaluating the safety and efficacy of CRISPR-edited CAR-T cells and PD-1 knockout T cells. The future of CRISPR in oncology lies in integrating it with combination therapies, multimodal editing approaches, and personalized treatment strategies informed by genomic and single-cell data, ultimately driving the transition towards smarter, safer, and more precise cancer therapeutics.
Journal
Med Research
Method of Research
Literature review
Subject of Research
People
Article Title
CRISPR Enabled Precision Oncology: From Gene Editing to Tumor Microenvironment Remodeling
Article Publication Date
5-Nov-2025