Table 1.

CRISPR-mediated modification of AR signaling in prostate cancer

Target gene	Study type	Cell line	Vector	Screening/verification	Experimental data	Ref
AR	In vitro	LNCaP	Lentiviral	PCR, CCK-8 assay, annexin V apoptosis assay	Reduced cell proliferation due to enhanced apoptosis	[54]
		LNCaP, R1-AD1	Plasmid	QPCR, sequencing, western blotting	High levels of tumor-specific AR variants and resistance to endocrine treatments in cell lines with AR gene rearrangements	[86]
		PacMet-UT1	Lentiviral	T7 endonuclease I assay, western blotting	Increased activity in the FGF and MAPK pathways	[87]
		C4-2, CWR22Rv1	Plasmid	Western blotting, qRT-PCR	Decreased cell proliferation	[88]
		LN-95	Plasmid	FACS, genotyping, western blotting, RNA-seq, IHC	Not necessarily leading to neuroendocrine differentiation	[89]
	In vitro, in vivo	LNCaP	Plasmid	IHC, RNA-Seq, qRT-PCR, luciferase assay, clonal and clonogenic assays, BrdU incorporation assays	Distinctive biological and tumorigenic characteristics, showing contrasting responses to enzalutamide as a therapeutic target	[90]
AR, NKX3-1	In vitro	LNCaP	Lentiviral	QPCR	Validation of the regulatory roles of specific enhancers in the expression of NKX3-1 and AR genes	[91]
AR, FABP5	In vitro	PC3-M, DU145, 22RV1	CRISPR/Cas9	Flow cytometry, western blotting, PCR, DNA sequencing, RNA sequencing	Inhibition of malignant cell characteristics, partly by disrupting the VEGF signaling pathway	[92]
AR-FL	In vitro	CWR22Rv1	Plasmid	Nucleofection, SURVEYOR assay, PCR and sequencing, FACS, western blotting, qPCR, RNA sequencing	AR variants drive cell growth and androgenic gene expression independently of FL-AR loss	[93]
AR-FL, AR-V7	In vitro	LNCaP-95	Plasmid	FACS, western blotting, genotyping, RNA sequencing, IHC	Identification of the necessity of AR-FL and AR-V7 in conferring resistance to abiraterone and enzalutamide	[94]
ERG	In vitro	Pten−/− R26-ERG organoids	Lentiviral	Histology, IHC, RNA-seq, ChIP-seq, ATAC-seq, proteomics, RIME	Significantly reduced AR-dependent gene expression	[95]
TMPRSS2-ERG	In vitro	Mouse prostate organoids	psCas9 plasmid	Puromycin selection, qPCR	Overexpression of ERG due to AR activity	[62]
GATA2	In vitro	22Rv1	Plasmid	Western blotting, qRT-PCR, single clone isolation, genotyping	Identification of GATA2 amplification’s role in enhancing TGFβ1 and AR signaling pathways	[96]
GREB1	In vitro, in vivo	LNCaP/AR	Lentiviral	Single-cell cloning, flowcytometry	Restoration of enzalutamide sensitivity in cells with high AR output	[97]
TLE3	In vitro	LNCaPCWR-R1, 22Rv1, LAPC4	Lentiviral	Real-time live cell proliferation monitoring, colony formation assays, RNA-seq, ChIP-seq, ChIP-qPCR, western blotting, (IHC), qPCR	Increased resistance to AR inhibitors, including apalutamide and enzalutamide	[98]
DOT1L	In vitro	LNCaP, PC3	Lentiviral, plasmid	QRT-PCR, RNA-seq, Microarray, ChIP, PCR, LC–MS/MS	Reduced viability of AR-positive prostate cancer cells	[99]
SF3B2	In vitro	22Rv1, LNCaP95	Plasmid	Western blotting, PAR-CLIP	Identification of SF3B2 as a key factor in the expression of AR-V7	[100]
TGM2	In vitro	PC3	Plasmid	Sequencing, western blotting	Increased AR transcription and decreased MUC1 expression	[101]
LCMT1	In vitro	HAP1, LNCaP, VCaP, LNCaP-AR, LAPC4	lentiviral	Immunoblotting	Enhanced AR activity, promoting the growth of castration-resistant prostate cancer	[102]
PARP-1, PARP-2	In vitro, in vivo	LNCaP	Lentiviral, plasmid	ChIP assay, RNA-seq, ChIP-seq, cell viability, soft agar colony formation assay	Selective inhibition of PARP-2 disrupts its interaction with FOXA1, leading to reduced AR-mediated gene expression and inhibited growth of AR-positive prostate cancer	[103]
IP6K2, XPO4, DRG1, PRKAB1, RP2	In vitro	C4, LNCaP, PC3, DU145, LAPC-4	Lentiviral, plasmid	Sanger sequencing, RT-qPCR, dose-response assays, AlamarBlue assay, quant-seq analysis	Altered response to enzalutamide following IP6K2 and XPO4 knockout due to deregulation of AR, mTORC1, and E2F signaling pathways	[104]
AREM1, AREM2	In vitro	C4-2B	CISPR/Cas9	Flow-sorting, qPCR, ChIP-qPCR, ChIP-seq	Significant decrease in AR mRNA levels	[105]
hnRNP A1	In vitro	CWR22Rv1	Plasmid	Puromycin selection, FACS, genomic PCR, western blotting	Increased expression of AR3	[106]
CAMKK2	In vitro	LNCaP, 22Rv1	Lentiviral, plasmid	Western blotting, immunofluorescence microscopy	Demonstrating that AR can utilize the CAMKK2-AMPK-ULK1 signaling pathway to stimulate prostate cancer by enhancing autophagy	[107]
		LNCaP	Plasmid	Western blotting, qRT-PCR	Decreased expression of two important lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase	[108]
	In vitro, in vivo	C4-2	Lentiviral, plasmid	Western blotting	Impaired tumor growth	[109]
SOX2	In vitro	CWR22-R1	CRISPR/Cas9	Western blotting	Modified gene expression profiles, particularly in pathways associated with resistance to AR antagonists	[110]
YAP1	In vitro	LNCaP	Plasmid	Western blotting, qRT-PCR, immunofluorescence and microscopy	Revealing that androgen differentially regulates YAP1-dependent gene expression	[111]
G3BP1	In vitro	LNCaP, 22Rv1	Lentiviral, plasmid	Immunoblot, CellTiter96, xenograft studies, RNA-seq, qRT-PCR	Revealing a G3BP1-SPOP ubiquitin signaling axis that promotes PCa progression through activating AR signaling	[112]
EZH2	In vitro	16DCRPC	Plasmid	GeneArt genomic cleavage detection, FACS, western blotting, sanger sequencing	Establishing a collaborative role for AR and EZH2 in promoting drug resistance	[113]
	In vitro, in vivo	PC-3	Plasmid	SURVEYOR assay, western blotting, MTT assay, wound healing and proliferation assays, qRT-PCR, annexin V-PI assay	Decreased H3K27me3 levels and increased apoptosis	[114]
EZH2, SETD2	In vitro, in vivo	C4-2, LNCaP	PX330 plasmid	QRT-PCR	Demonstrating the role of the SETD2-EZH2 axis in linking metabolic and epigenetic signaling pathways to suppress prostate cancer metastasis	[115]
PSA	In vitro	LNCap, PC3, AT3B-1, DU145, RWPE1	Plasmid	Luciferase assay, qRT-PCR, MTT assay, colony formation assay, cell migration assay, transwell migration assay, annexin V-PI assay	Reduced cell proliferation and migration, enhanced apoptosis	[56]
Survivin	In vitro	PC3	Plasmid	Colony PCR, sequencing, qRT-PCR, XTT assay, annexin V-PE/7- AAD assay, CCK-8 assay	Induced apoptosis and downregulation of FBXO5 and RRM2	[59]
SPOP	In vitro	LNCaP,	Plasmid	Genomic DNA and cDNA sequencing	Elevated GLI3 protein levels, indicating decreased degradation	[116]
	In vitro, in vivo	DU145	Lentiviral	Western blotting, real-time quantitative PCR, IP, in vivo xenograft assay	Identifying SPOP as a tumor suppressor that promotes the ubiquitination and degradation of NANOG	[117]
SPOP, Caprin1	In vitro	293T, LNCaP, 22Rv1, PC-3, DU-145, C4-2	PX459 plasmid	Western blot, sanger sequencing, cell cycle distribution and cell death, qRTPCR, EdU incorporation assay, immunofluorescence, migration assay	Resistance to cell death induced by stress granule inducers (e.g., docetaxel, sodium arsenite, and H2O2)	[118]
SPOP, HIPK2	In vitro, in vivo	PC-3, DU145	PX459 plasmid	Western blot, sanger sequencing, CCK-8 assay, colony formation assay	High levels of genomic instability	[119]
SPOP, SQSTM1/p62, ATG3, ATG5, ATG7, NFE2L2	In vitro	PC-3, DU145	PX459 plasmid	Western blotting, sanger sequencing	Increased autophagy and activation of Nrf2	[120]
FOXA1	In vitro	LNCaP	Plasmid	Western blotting, dot blotting	Altered expression of Casp-9, Bax, CCND1, CDK4, and fibronectin; no changes observed in Casp-3, Bcl-2, survivin, β-catenin, c-Myc, and GSK-3B; inhibition of CCND1 protein expression in LNCaP cells	[121]
		LNCaP, 22Rv1	Lentiviral, plasmid	Western blotting, cell proliferation assay, luciferase reporter assay, allele-specific ChIP-qPCR	Reduction in FOXA1 expression, leading to decreased cell growth	[122]
		LNCaP, 22RV1	Lentiviral, plasmid	Sanger sequencing, ChIP-seq	Reaffirming FOXA1 central role in mediating androgen receptor-driven oncogenesis	[123]
		LNCaP, LNCaP 42D, LNCaP 42 F	Lentiviral	Immunoblot, western blotting, immunohistochemistry	Inhibited cell proliferation	[124]
		LNCaP	Lentiviral	Puromycin selection, PCR, qRT-PCR, sanger sequencing	Upregulation of TGFB3, a gene encoding a ligand in the TGF-β pathway	[125]
	In vitro, in vivo	Mouse prostate organoids, Rosa26-Cas9 organoids	Lentiviral, plasmid	Western blotting, RNA-seq, ATAC-seq, ChIP-seq	Disruption of normal luminal epithelial differentiation programs	[126]
FOXA1, FOXP1, PTEN	In vivo	MEF cells from LSL-Cas9 mice, HEK293T	AAV-plasmid	Histochemical analysis, PCR	Induction of epithelial plasticity due to FOXA1 loss and increased cell proliferation due to FOXP1 loss	[127]
NANOG and NANOGP8	In vitro, in vivo	DU145	PX330 plasmid	PCR, genotyping, western blotting	Reduced malignant potential, diminished sphere formation, anchorage-independent growth, migration ability, and chemoresistance	[61]
SRD5α2	In vitro	DU145	A cationic liposome preparation carrying sgRNA on its surface	QRT-PCR	Discovery of a novel gene-editing approach targeting SRD5α2 to offer alternative treatments for prostate cancer without the adverse effects of current medications	[57]
CHD1	In vitro	22Rv1	CRISPR/Cas9	Immunoblotting, IHC, FISH, DNA sequencing	Improved response rate and extended efficacy during abiraterone treatment	[128]
	In vitro, in vivo	22Rv1, RWPE-1	Plasmid	FACS, SURVEYOR mutation assay, PCR, sanger sequencing	Increased vulnerability to DNA damage, reduced efficiency of error-free HR repair, and elevated reliance on error-prone NHEJ repair, leading to genomic instability	[129]
BRCA2	In vitro	LNCaP	Lentiviral	Western blotting	Heightened sensitivity to SPA, resulting in increased DNA damage and apoptosis	[130]
		LNCaP	Lentiviral	Sanger sequencing, qPCR	Elevated SRC phosphorylation and greater responsiveness to SRC inhibitors like dasatinib, bosutinib, and saracatinib	[131]
BRCA2, RB1	In vitro	LNCaP, 22RV1	Lentiviral	FISH, western blotting, qPCR, RNA sequencing	Induced epithelial-to-mesenchymal transition, leading to greater invasiveness and a more aggressive cancer phenotype	[132]
TP53, RB1	In vitro, in vivo	LNCaP	Lentiviral, plasmid	Western blotting	High proliferation rates, resistance to different therapies	[133]
HOXB13	In vitro	22Rv1, DU145, LNCaP, C4-2, PC3, RWPE-1, VCaP, C4-2B	Plasmid, CRISPR/Cas9 constructs (GFP expressing)	Western blotting, qPCR, immunofluorescence studies, MTT assay, BrdU assay, wound scratch assay	Induction of apoptosis and strong suppression of tumor cell proliferation and migration	[134]
		CWR22Rv, LAPC4	Lentiviral, plasmid	Western blotting	Altered cell proliferation patterns	[135]
CTCF	In vitro	22Rv1	Lentiviral, plasmid	PCR, sanger sequencing, western blotting	Reorganization of CTCF looping and changes in H3K27ac loops at the MYC region	[136]
SMYD2	In vitro	CWR-R1ca	Plasmid	Western blotting, wound-healing assay, colony formation assay, transwell assay	Decreased viability, reduced migration and invasion capacity, and fewer colonies formed	[137]
SYNCRIP	In vitro	LNCaP/AR, CWR22Pc, MDA-PCa-2b	Lentiviral, plasmid	CellTiter-Glo, flowcytometry, western blotting	Increased APOBEC-driven mutagenesis	[138]
p54/nrb (NONO)	In vitro, in vivo	CWR22Rv1	Plasmid	Western blotting, qRT-PCR, FACS, genomic PCR	Lowered PCGEM1 expression, leading to reduced tumor growth	[139]
ARNTL	In vitro, in vivo	LNCaP, LNCaP-42D, LNCaP-ResA	Lentiviral, plasmid	Western blotting, puromycin selection, cell viability and xenograft studies	Inhibited growth of prostate cancer cells	[140]
(N-terminal domain of) PIP5K1α	In vitro, in vivo	LNCaP C4-2, LNCaP C4-2 SG	CRISPR/Cas9	Sequencing	Reduced ability of cancer cells to grow and migrate	[141]
NKX3.1	In vivo	C57BL6, Swiss-Webster, B6D2F1 mice	PX330 plasmid	Genotyping, histologic analysis, in situ TUNEL staining	Allelic loss of Nkx3.1 led to decreased Nkx3.1 protein level and increased proliferation	[64]