Table 6.
CRISPR‐based HCC therapeutic strategies
| Therapeutic targets | Functions | In vitro or in vivo models | gRNA design | CRISPR manipulation | Methods and vectors | Gene‐editing efficiency (%) | Therapeutic effects | Ref. |
|---|---|---|---|---|---|---|---|---|
| ZIC2 | A transcription factor (TF) for self‐renewal maintenance of liver cancer stem cells (CSCs) | Hep3B cells, mouse models with tumor xenograft | 5’‐CCATCACCACTCCGCCGCGG‐3’, 5’‐TTCACGGTCCTGCATCTCGG‐3’ | Knockout | Cas9/sgRNA plasmid delivered by lentivirus (in vitro) and CRISPR‐engineered Hep3B cells delivered by e subcutaneous injection (in vivo) | Unshown | Inhibition of self‐renewal of liver cancer stem cells (CSCs) and tumor propagation | [ 25 ] |
| Aspartate β‐hydroxylase (ASPH) | A transmembrane protein member in α‐ketoglutarate‐dependent dioxygenase family | HepG2 cells | 5’‐ATGGAGGACACAAGAATGGG‐3’, 5’‐TAAACAGAGACAAAGCATGG‐3’, 5’‐CCTAGTACAAAATACGTGACGTAGAA‐3’ | Knockout | Cas9/sgRNA plasmid delivered by human immunodeficiency viruses (HIV) (in vitro) | Unshown | Inhibition of tumor growth and induction of tumor cell senescence | [ 166 ] |
| BAX and BCL2 | Related to the sensitivity of cells to apoptosis | HepG2 cells | BAX: sgRNA38, 5’‐GAGAACAGGGTACGATAACCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTCGTACACCATCAGGGTACGTCGTACCCTGTTCTCAGAGCGGAAGCGTGCTGGGCTCCGAACAGCGGAAGGTGGTTCGAAGCTGGGGCTTTGGACATAAGAGAACAGGTTTTTT‐3’; BCL2: sgRNA39, 5’‐ GACGGGACCAAACCTCCCGAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTGGTTTAATCAGAGTAGAGGAGCTGACTCCTTTGGTTGGACTAAGGTTTGGTCCCGTCAGAGCGGAAGCGTGCTGGGCTCCGAACAGCGGAAGGTGGTTCGAAGCTGGGGCTTTGGACATAAGACGGGACCTTTTTT‐3’ | CRISPRi for BCL and CRISPRa for BAX | MS2‐dCas9+sgRNA38 and Rev‐dCas9‐VP64+sgRNA39 plasmid delivered by lipofectamine (in vitro) | In vitro: up to 8‐fold activation and 80% repression | Induction of tumor cell apoptosis | [ 167 ] |
| CXC chemokine receptor 4 (CXCR4) | A specific receptor of chemokine stromal cell‐derived factor‐1 (CXCL12) with a strong chemotaxis effect on lymphocytes | HepG2 cells, mouse models with tumor xenograft | 5’‐CACCGGGCAATGGATTGGTCATCC‐3’ | Knockout | Cas9/sgRNA plasmid delivered by lipofectamine (in vitro) and CRISPR‐engineered HepG2 cells delivered by e subcutaneous injection (in vivo) | In vitro: 29.5% | Nearly 50% CXCR4 downregulation, inhibition of tumor proliferation, migration, invasion the malignancy, reversion of epithelial‐mesenchymal transition (EMT), increased chemosensitivity to the antitumor drug cisplatin | [ 168 ] |
| Euchromatic histone‐lysine N‐methyltransferase 2 (EHMT2), also known as G9a | A lysine methyltransferase to di‐methylate lysine 9 of histone H3 (H3K9me2) | BEL‐7402 cells, SMMC‐7721 cells, THLE‐3 cells, mouse models with orthotopic tumor implantation | 5’‐GGGTCACTTCTCCTGAACGC‐3’, 5’‐GGTCACTTCTCCTGAACGCC‐3’ | Knockout | PX459 delivered by lentivirus (in vitro) and CRISPR‐engineered BEL‐7402 cells delivered by e subcutaneous injection (in vivo) | Unshown | Inhibition of the proliferation and migration of HCC cells in vitro, inhibition of HCC tumorigenicity in vivo | [ 169 ] |
| Granulin (GRN) | A potent pluripotent mitogen and growth factor maintaining self‐renewal of liver CSCs | Hep3B cells |
5’‐TAGAGATGATAGCGCGTGTCTGG‐3’, 5’‐GGCGCCTGCAGGATGGGTTAAGG‐3’ 5’‐TTGGAGAATCATGTGACGTCGG‐3’ 5’‐GATCCCTAGAAATGGGGTGTGG‐3’ |
CRISPRi | dCas9‐suppressor plasmid and gRNA plasmid delivered by lipofectamine (in vitro) | In vitro: about 80% | Inhibition of proliferation and invasion up to fourfold | [ 43 ] |
| Glutaminase 1 (GLS1) | An enzyme converting glutamine to glutamate, which is highly expressed in HCC | LO2 cells, SMMC‐772 1cells, HCCLM3 cells, Hep3B cells, mouse models with tumor xenograft | Unshown | Knockout | Cas9/sgRNA plasmid delivered by lentivirus (in vitro) and CRISPR‐engineered HCCLM3 and SMMC‐7721 cells delivered by e subcutaneous injection (in vivo) | Unshown | Decrease of stemness‐related genes expressing, inhibition of CSC properties, and tumorigenicity | [ 170 ] |
| CCAAT/enhancer‐binding protein‐beta (C/EBPβ) | A recurrent hypomethylated enhancer related to poorer HCC prognosis | LO2 cells, BEL‐7404 cells, Hep3B cells, HepG2 cells, Huh7 cells, PLC5 cells, SK‐Hep1 cells, mouse models with tumor xenograft | 5’‐CACACACACAGGGCCACCGA‐3’ | Knockout | Cas9/sgRNA plasmid delivered by jetPRIME (in vitro) and CRISPR‐engineered HCC cell lines delivered by e subcutaneous injection (in vivo) | Unshown | Inhibition of driver oncogenes and tumorigenicity | [ 171 ] |
| Nuclear receptor binding SET domain‐containing protein 1 (NSD1) | Involving in tumorigenesis via regulating Wnt/β‐catenin signaling pathway | Huh7 cells, Hep3B cells, SMMC‐7721 cells, HepG2 cells, SK‐Hep1 cells, mouse models with tumor xenograft | 5’‐TTGGATTGACCATTACCGAA‐3’, 5’‐TGGATTGACCATTACCGAAA‐3’, 5’‐GCAAGTGCTGTAGGACCACC‐3’ | Knockout | Cas9/sgRNA plasmid delivered by lentivirus (in vitro) and CRISPR‐engineered HCC cell lines delivered by e subcutaneous injection (in vivo) | Unshown | Inhibition of tumor proliferation, migration, invasion | [ 172 ] |
| Zinc‐finger protein 384 (ZNF 384) | Promoting tumor growth by upregulating Cyclin D1 expression | Huh7 cells | 5’‐CACCGGCCTCAGTGTCCCTGCCCTC‐3’, 5’‐CACCGGCCAGAGAAGGGCTGTGGTC‐3’ | Knockout | wPGL3 plasmid delivered by lentivirus (in vitro) | Unshown | Inhibition of tumor proliferation via inhibition of Cyclin D1 | [ 173 ] |
| lncRNA‐RP11‐156p1.3 | Belonging to HCC‐associated lncRNA network | HepG2 cells | 5’‐GCCGGGGAGCAGGGTGCGCCGGG‐3’, 5’‐ACGACGACGTAGGATGCGCCAAA‐3’ | Knockout | RNP delivered by CRISPRMAX | Unshown | Significant decrease of cell viability, TNF α and NFκβ protein levels | [ 174 ] |
| Epidermal growth factor receptor (EGFR) | A transmembrane receptor‐associated with the growth and proliferation of HCC | HepG2 and Huh7 cells, H22 cells‐bearing mice | 5’‐GCATGGCGCCGTGCGGTTCA‐3’, 5’‐AGTAACAAGCTCACGCAGTT‐3’ | Knockout and combing with sorafenib | PX458 delivered by an aptamer‐coated hollow mesoporous silica nanoparticle (in vitro and in vivo) |
In vitro: 66.3% In vivo: unshown |
Efficient in vitro EGFR‐editing and in vivo gene therapy for tumor inhibition as well as good synergistic drug therapy | [ 110 ] |
| Survivin (BIRC5) | Directly mediating tumor recurrence and metastasis | BEL‐7402 cells, BEL‐7402 cells‐bearing mice | 5’‐TCTTGAATGTAGAGATGCGG‐3’ | Knockout and combing with sorafenib | Cas9/sgRNA plasmid delivered by a lactose‐derived branched cationic biopolymer (LBP) (in vitro and in vivo) |
In vitro: 21.3% In vivo: 26.4% |
Efficient in vitro editing and in vivo HCC therapy | [ 104 ] |
| WNT10B | A member of the Wnt family encoding secreted proteins | HepG2 cells and HepG2 cells‐bearing mice | 5’‐TCTTGGTTCCCAGGGCTCTA‐3’, 5′‐ GCCTCCGCTCAGCTTAATCT‐3’ | Knockout | RNP delivered by cell‐selective extracellular vesicle (in vitro, ex vivo, and in vivo) |
In vitro: about 30% Ex vivo: unshown In vivo: unshown |
Decreased the protein expression of WNT10B and tumor inhibition in vitro, ex vivo, and in vivo | [ 112 ] |