Melanoma is the most deadly skin cancer, and the 5-years survival rate of patients with stage IV melanoma is below 15% (Fleming et al., 2015). The incidence of invasive melanoma is still growing faster than any other cancer (Prado et al., 2019), accounting for about 70% of skin-cancer-related deaths (Leonardi et al., 2018). The main cause of death from melanoma is tumor metastasis (Fidler, 2015). In recent years, studies found that the incidence, prognosis, and treatment of melanoma are closely related to CKIT, NRAS, BRAF, and other gene mutations (Nassar and Tan, 2020; Pham et al., 2020). Patients with gene mutation have a poor prognosis, are prone to relapse, metastasis, and other malignant events (Rastrelli et al., 2014; Yde et al., 2018).

Because melanoma is a malignant tumor of the skin, the in-situ tumor model can be established through a relatively simple subcutaneous seed tumor. Hence, numerous experiments exploited melanoma as the research object. Deng et al. used poly (β-amino ester) copolymer nanoparticles to carry SpCas9/sgRNA plasmids targeting CDK5 to achieve NHEJ-mediated destruction (Deng et al., 2020). Compared with the PEI 25K and HP transfection reagent, these showed superior transfection efficiency in B16F10 cells (Figure 2A). By effectively knocking out Cdk5 target gene, PD-L1 expression was down-regulated in vivo, the CTL mediated immune response was restored, and the immunosuppressive tumor microenvironment was reversed (Figure 2B). Tumor growth was inhibited in B16F10 tumor-bearing mice (Figure 2C). Compared with the anti-PD-L1 antibody, it is capable of maintaining a relatively long-term therapeutic effect. Zhang et al. developed a double-locked nanoparticle called DLNP that can stably exist in blood circulation or normal tissues and release the CRISPR/Cas13a system in the tumor microenvironment with low pH and high H₂O₂ concentration. It promotes cellular internalization of the CRISPR/Cas13a system and activation of gene editing after entry into the tumor tissue (Zhang et al., 2019). Improved gene editing efficiency at tumor sites and reduced side effects caused by unintended activation of CRISPR/Cas13a in normal tissue were observed. By systemic administration, the tumor growth of B16F10 tumor-bearing mice was significantly inhibited, and the survival rate was improved.

Plk1 (Polo-like kinase 1) belongs to the Polo-like kinase family, which is a serine/threonine kinase abundant in eukaryotic cells. Overexpression of Plk1 has been found in numerous tumor tissues and model tumor cells (such as A375 cells), and inhibition of Plk1 expression can lead to apoptosis of tumor cells, providing a good strategy for tumor therapy (Lee et al., 2015; Gutteridge et al., 2016; Iliaki et al., 2021). Wang et al. prepared a Cas9 protein/sgPlk1 plasmid carrier with gold nanoclusters as the core and achieved 26.2% Plk1 genome modification in vitro, which is more than 10 times more effective than the traditional plasmid transfection method (Wang et al., 2017). The in vivo antitumor effect of granulosa was evaluated on the A375 nude mouse subcutaneous tumor model. Compared with other control groups, the nanoparticles had lower toxicity and the most significant inhibitory effect on tumor growth.

Photothermal therapy is a treatment method using materials with high photothermal conversion efficiency, implanting them inside the body, gathering them near the tumor tissue with targeted recognition technology, and converting the light energy into heat energy under the irradiation trigger of an external light source (usually near infrared light) to kill cancer cells (Melamed et al., 2015). The advantage of this therapy over traditional cancer therapies is that effective treatments can be performed with precision and few side effects. Numerous studies have addressed this method’s tumor inhibition through photothermal therapy and the interaction of nanoparticles (Hirsch et al., 2003; Dickerson et al., 2008; Chen et al., 2010; Day et al., 2011; Day et al., 2012). Kim et al. constructed a metal-lipid hybrid nanoparticle (MLN) to deliver plasmid DNA of sgRNA and Cas9 proteins encoding TGF-β (Kim et al., 2021). In B16F10 tumor-bearing mice, ifn -γ, cytotoxic T-cells, and mature dendritic cells were increased in the tumor microenvironment by intratumoral injection of the nano-complex plus Near Infra-Red (NIR) irradiation (Figure 3B), which could ablate the primary tumor (Figure 3A) and prevent distant growth of secondary B16F10 cells and lung metastasis (Figure 3C).

Breast cancer is the leading cause of cancer in women, which mostly occurs over 40 years old (Radecka and Litwiniuk, 2016). The incidence of breast cancer is related to genetic and environmental factors (Peairs et al., 2017; Barzaman et al., 2020). According to histological characteristics, breast cancer can be divided into human epidermal growth factor receptor two overexpression (HER2+), hormone receptor positive and three negative breast cancer (TNBC) (Nagini, 2017). Among breast cancers, TNBC is a special breast cancer subtype characterized by deletion of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Foulkes et al., 2010). TNBC lacks effective targeted therapy (Cardoso et al., 2018), is highly invasive and metastatic, and has the highest mortality rate among all breast cancer subtypes (Dent et al., 2007; Foulkes et al., 2010). In recent years, nanomaterials combined with gene therapy has gradually become the focus of TNBC treatment, Chen et al. designed an intelligent nanocomposite that can release CCL25 protein and CD47 siRNA in the tumor tissue of TNBC mouse model. They proved that the transmission of ccl25 in tumor can promote the tumor infiltration of CCR9 + CD8 + T cells by blocking CD47/Sirpα and PD-1/PD-L1 signaling pathways, so as to significantly inhibit tumor growth (Chen et al., 2020). Guo et al. reported a nanolipogel with tumor-targeting, deformable, and non-cationic characteristics, called tNLG, which was used to edit the CRISPR genome in TNBC tumors and successfully inhibited the expression of breast cancer gene Lcn2, mediating a gene editing efficiency of more than 81% (Guo et al., 2019). Deletion of the Lcn2 gene significantly inhibited the migration and mesenchymal phenotype of TNBC cells, thus attenuating the aggressive spread of TNBC. In the TNBC in situ tumor model, administration of MDA-MB-231 tumor-bearing mice through the tail vein inhibited 77% of TNBC tumor growth with minimal systemic toxicity. Deng et al. (Fidler, 2015) reported that poly (β -amino esterification) copolymer nanoparticles coated with CDK5-targeting SpCas9/sgRNA plasmids showed superior transfection efficiency in 4T1 cells. (Deng et al., 2020). Compared with the anti-PD-L1 antibody group, pNDA group and PBS group, the tumor volume and weight were significantly reduced in the TNBC lung metastasis model, and the occurrence of lung metastasis was decreased in the nanoparticle group (Figure 4A). Immunohistochemical results showed that the infiltration of CD8⁺ T-cells was high, and the expression of Cdk5 was decreased. TUNEL staining also showed enhanced apoptosis of tumor cells after nanoparticle treatment (Figure 4B).

Lung cancer is one of the most threatening malignant tumors threatening human health and survival (Mao et al., 2016; de Sousa and Carvalho, 2018). In the recent 50 years, the incidence and mortality of lung cancer have been reported in numerous countries, and it has been found to rank in the top two cancer types for both men and women (Romaszko and Doboszyńska, 2018). Surgical treatment is suitable for early stage of lung cancer (Hoy et al., 2019). A study on lung cancer patients showed that tumor suppressor gene mutations and the overexpression of oncogenes may be related to the occurrence of lung cancer (Liu et al., 2004). In recent years, gene editing techniques have been used for in vitro experiment of cancer (Jubair and McMillan, 2017). He et al. constructed a natural nanopolymer by combining hyaluronic acid (AHA) functionalization with aptamer (AS1411) and hyaluronic acid (PHA) functionalization with peptide (TAT-NLS) to deliver CRISPR-Cas9 plasmids specifically to tumor cells and achieve effective gene editing. The CTNNB1 gene encoding β -catenin was knocked out, and PD-L1 expression was down-regulated in tumor cells, successfully reversing tumor immunosuppression and immune escape of H1299 (He et al., 2020). Gene-edited tumor cells effectively enhance T cell immunity, including proliferation, cytokine release, and cytolysis activity.

Photothermal therapy (PTT) has attracted increasing attention in the field of tumor therapy owing to its spatio-temporal controlled mode and non-invasive nature (Song et al., 2018). Pan et al. designed a nanocomposite. CRISPR-Cas9 was covalent anchoring of photodegradable 4- (hydroxymethyl) -3-nitrobenzoic acid (ONA) molecules on lanthanum-doped conversion nanoparticles (UCNP), and then coated with polyethylene imine (PEI) to assist the escape of endosomes (Pan et al., 2019). The nano-complex can be effectively internalized by cells via the endocytosis pathway, followed by endosomal escape and cytoplasmic release of ucNPS-Cas9 loaded in the cytoplasm. When exposed to NIR, UCNP emit local ultraviolet light and trigger the rupture of the junction. Consequently, Cas9 can be released from the surface of the UCNP and thus enter the nucleus for gene editing. Tumor growth was successfully inhibited in xenografted nude mouse models of A549 cells by targeting oncogene markers (PLK-1 gene). Although PTT can increase the local temperature of the body to over 50°C and kill tumor cells, it can also cause damage to normal tissues and carry the risk of recurrence or metastasis (Gao et al., 2019). Li et al. applied a low oxygen responsive gold nanorods that can carry sgRNA targeting HSP90α (Li et al., 2021). Due to the hypoxic state of the tumor microenvironment, the azo group of the nano-complex is selectively reduced by overexpression reductase, resulting in the release of Cas9 and subsequent HSP90α gene knockout, which reduces the thermal resistance of cancer cells. Under mild PTT conditions, the nano-complex can achieve efficient tumor ablation in vivo and in vitro. This also reduces thermal damage to normal surrounding tissues.

Hepatocellular carcinoma (HCC) is the third most common cancer worldwide. Surgical resection and liver transplantation are considered to be the only treatments, but are often limited by low liver function and a shortage of liver donors (Waghray et al., 2015; Valverde-López et al., 2018). The epidermal growth factor receptor (EGFR) is a transmembrane receptor that plays an important role in various tumors, especially liver cancer, leading to the growth and proliferation of tumor cells. Sorafenib, a chemotherapy drug, assumes an anti-tumor role by specifically inhibiting multiple molecular targets, such as EGFR and VEGFR2 (Redd Bowman et al., 2020). Zhang et al. designed a polyaminoamine aptamer coated hollow mesoporous silica nanoparticle, which has good stability in blood circulation and a large drug load, and can jointly deliver the Sora and CRISPR/Cas9 system (Zhang et al., 2020). Apt-modified NPs surfaces can effectively enhance the uptake of NPs by HCC cells, thus reducing the side effects of Sora. Moreover, the CRISPR/Cas9 system co-delivered with Sora synergistically inhibits the expression of EGFR and downstream PIK3 Akt pathways, with no detectable off-target effects and a powerful anti-angiogenesis effect. The synergistic efficacy of nanocomposites was studied both in vitro and in vivo. Both model systems demonstrated that NP has strong cytotoxicity to HCC cells by specifically binding EpCAM receptors on tumor cell membranes. In vivo fluorescence imaging shows the accumulation of NPs in the tumor area, while HE staining and blood biochemical analysis showed no significant damage to major organs. Furthermore, effective gene editing of EGFR in vivo was confirmed by sequencing, and inhibition of EGFR expression in tumor tissues was detected by IHC.

The PLK1 gene has also been targeted in related studies of liver cancer. Li et al. constructed a proprietary ionizable lipid nanoparticle named iLP181 (Li et al., 2022). Four plasmids containing PLK1-targeting Cas9 protein and sgRNA were designed. PsgPLK1 with the best activity was selected and loaded with iLP181. Studies have shown that iLP181/psgPLK1 is effectively internalized by hepatocellular carcinoma cells by binding ApoE, resulting in long-term in vivo and in vitro gene editing and significantly inhibiting tumor growth in HepG2-LuC-bearing mice. Compared with Lipo2000 on the market, iLP181 shows strong endogenous escape when delivering nucleic acid.

Glioblastoma (GBM) is the most common and fatal primary brain tumor in adults. The mean survival was only 12–14 months, even after a combination of treatments including surgery, chemotherapy and/or radiation (Davis, 2016). In primary glioblastoma, the molecular changes are mainly the expansion and overexpression of EGFR (Eskilsson et al., 2018), while in secondary glioblastoma, the molecular changes are mainly p53 mutation (Wirsching et al., 2016). BBB block more than 98% of substances from entering the central nervous system and also limit the passage of drugs, including therapeutic drugs. The application of nanocarriers increases the possibility of drugs, nucleic acids, etc. crossing the BBB and further treatment. Yang et al. designed a lipid polymer hybrid nanoparticle (LphNS-CRGD) for efficient and targeted delivery of CRISPR/Cas9 plasmids targeting temozolomide (TMZ) resistance gene O6-methylguanine DNA methyltransferase (MGMT) (Yang et al., 2021). LPHNS-cRGD can target GBM cells and mediate the transfection of pCas9/MGMT to down-regulate the expression of MGMT, resulting in increased sensitivity of GBM cells to TMZ. In vivo, local FUS irradiation can safely increase BBB permeability and allow nanoparticles to accumulate in tumors of in situ tumor-bearing mice, enhancing the therapeutic effect of TMZ on glioblastoma, inhibiting tumor growth, and prolongating survival of tumor-bearing mice with high biosafety. Co-delivery of Cas9 mRNA and sgRNA using delivery system is a promising strategy to efficiently edit the genome in cells. Kataoka et al. developed a PEGylated polyplex micelle (PM) to co-encapsulate the Cas9 mRNA and sgRNA for genome editing, which could prevent the sgRNA release upon dilution and enhance the tolerability of cas9 mRNA and sgRNA against enzymatic degradation (Uchida and Kataoka, 2019). They further achieved effective genome editing in the mouse brain parenchyma in vivo using this PM with co-encapsulated Cas9 mRNA and sgRNA (Abbasi et al., 2021). Rosenblum et al. reported a liposome nanoparticle equipped with PLK1-targeted Cas9 mRNA and sgRNA gene-editing tools, and the gene-editing rate of this system was up to 98% in vitro in a variety of cancer cell types (Figure 5A) (Rosenblum et al., 2020). single-dose administration of nanoparticles on tumors in a mouse GBM model resulted in approximately 70% PLK1 gene editing. The induction of apoptosis in vivo was assessed by activated Caspase three staining, and increased median survival by approximately 50% and overall survival by 30% in GBM tumor-bearing mice (Figure 5B). Liu et al. synthesized a nanoparticle carrying sgRNAs (Liu et al., 2019). PLys and Cas9/sgRNA complexes make up the core, as well as mPEG, and the core and shell are connected by 2,5-dihydro-2,5-dioxofuran-3-acetic acid (CA). Due to the degradation of CA in acidic environment, the complex exhibits an acid response. In the acidic tumor microenvironment, the mPEG shell can be peeled off, and the inclusion can be efficiently accumulated in the tumor, resulting in gene editing. Tumor growth can be inhibited in heterogeneous tumor models with two cell subpopulations by carrying sgRNAs targeting STAT3 and RUNX1.

Every year, there are more than 500,000 new cases of cervical cancer worldwide. Infection with human papillomavirus (HPV) causes 90% of cervical cancer cases (Hu and Ma, 2018; Okunade, 2020). HPV is a spherical DNA virus that causes proliferation of the squamous epithelium, the mucous membrane of the human skin. More than 130 species of HPV have been isolated so far. Among them, HPV-16 and HPV-18 are the main drivers of malignant transformation of cervical epithelial cells. To date, treatment outcomes for cervical cancer remain unsatisfactory, with more than 30% of patients initially treated diagnosed with recurrence and metastasis within 2 years and a 5-years survival rate of less than 10% (Pimple and Mishra, 2019). Therefore, it is urgent to develop new strategies to improve the therapeutic treatments of cervical cancer. Zhen et al. developed a novel liposome nanoparticle containing CRISPR/Cas9 gene-editing tool, which can inhibit the proliferation of HPV16-positive cervical cancer SiHa cells and induce apoptosis by inactivating the hr-HPV16E/E7 oncogene (Zhen et al., 2020). Injection of cationic liposomes targeting HPV16 E6/E7 into subcutaneous tumor models in nude mice significantly inhibited tumor growth without significant toxicity.

Gold nanoclusters have good biocompatibility, chemical inertia, strong fluorescence emission, and tunable surface functionalization, which can efficiently complete the delivery task, and are widely used in tumor and other disease models (Vankayala et al., 2015). Tao et al. designed a nanocarrier composed of protamine with AuNC, a gold nanocluster with high biocompatibility, for transporting Cas9 — sgRNA plasmids for genome editing (Tao et al., 2021). The nanocomplex can effectively knock out the oncogenic E7 gene and inhibit the proliferation of HeLa cancer cells. It also can effectively achieve genome destruction in different cancer cells, which significantly broadens its further applications in cancer treatment. In addition, protamine-AuNCs have excellent fluorescence properties, indicating great potential of our nanocarriers for imaging tracking. Ju et al. also proved that AuNCs could be assembled with purified Streptococcus pyogenes Cas9 (SpCas9) protein under physiological conditions (Ju et al., 2019). The complex is stable at higher pH and decomposes at lower pH. Due to the low pH microenvironment of the tumor, the assembly-decomposition process can promote the entry of SpCas9 into the tumor nucleus and perform its cleavage function. SpCas9—AuNCs combined with HPV18 E6 sgRNA—effectively knocks out the oncogenic E6 gene, triggering the expression of tumor suppressor protein p53, restoring its function, and inducing the apoptosis of cervical cancer cells. Importantly, the process had little effect on other human cells without the HPV E6 gene, demonstrating the high efficiency and specificity of gene therapy for cancer.

Globally, ovarian cancer is the seventh most common cancer in women, with a 5-year survival rate of less than 45%. About 140,000 women die of ovarian cancer every year (Webb and Jordan, 2017; Penny, 2020). Because ovarian cancer lacks specific symptoms in its early stages, and tests do not always yield positive results, most ovarian cancer is not diagnosed until later in life, and many patients have developed malignant peritoneal effusion (Roett and Evans, 2009; Aleksandra Kujawa and Lisowska, 2015). Rosenblum et al. reported a lipid nanoparticle loaded with PLK1 targeted Cas9 mRNA and sgRNA gene-editing tools (Rosenblum et al., 2020). They created a mouse model of peritoneal disseminated ovarian cancer, OV8-Mcherry, and injected nanoparticles through the abdominal cavity. The results showed that the nanoparticles could be selectively ingested into disseminated ovarian tumors (Figures 6A,C), and the gene editing rate was up to 80% in vivo (Figure 6B), and the tumor growth was inhibited (Figure 6D), and the survival rate of tumor bearing mice was increased by about 80% (Figure 6E).

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous group of malignant tumors, which is usually caused by drinking and smoking, includes oropharyngeal squamous cell carcinoma (OPSCC), laryngeal squamous cell carcinoma, and oral squamous cell carcinoma (Johnson et al., 2020; von Witzleben et al., 2020). Surgical treatment can lead to visible and functional facial deformities: radiation and chemotherapy are less specific for individual tumors, and have systemic toxic side effects (Murphy et al., 2007). Local or regional metastasis occurs in some patients, which reduces the survival rate to less than 1 year (Schwartz et al., 2000; Forastiere et al., 2001). HuR is an RNA-binding protein encoded by ELAVL1 gene, which plays an important role in regulating the survival, metastasis and drug resistance of HNSCC. Wang et al. designed two different liposome nanocarriers, namely SLN-HPR and LIP-HPR, to deliver CRISPR/Cas9 system and epubicin (Epi), respectively (Wang et al., 2021). These nanoparticles have numerous effects: 1) promoting endosome escape and nuclear localization through the pH response and nucleo-targeted H and R polypeptide sequences; 2) maintaining blood circulation through PEG modified nanoparticles and passively targeting tumors through enhanced permeability and retention effect (EPR); 3) EGFR targeting and cell internalization can be improved by endocytosis mediated by EGFR ligand P; 4) nuclear and/or cytoplasmic release of Epi and/or CRISPR/CAS-9 as a topoisomerase inhibitor HuR knockout system inhibits tumor progression and multiple survival and metastasis pathways, and regulates drug resistance. By establishing a SAS/LUC-BEARING mouse model, HuR CRISPR/SLN-HPR pre-knocked out HuR in SAS cells and co-treated SAS/LUC mice with Epi/LIP-HPR, achieving the most significant antitumor effect.