CRISPR-Cas9 system in autosomal dominant polycystic kidney disease: a comprehensive review

CRISPR-Cas9 system in ADPKD

Article information

Child Kidney Dis. 2025;29(1):4-11

Publication date (electronic) : 2025 February 28

doi : https://doi.org/10.3339/ckd.25.008

Seungyeon Kang ¹^,^*

, Se Jin Park ²^,^*

, Min Ho Lee ³

, Andreas Kronbichler ⁴^,⁵

, Jae Il Shin ⁶^,⁷

¹Department of Pharmacology and Toxicology, Molecular Genetics and Microbiology, University of Toronto, Toronto, ON, Canada

²Department of Pediatrics, Changwon Hanmaeum Hospital, Hanyang University College of Medicine, Changwon, Republic of Korea

³Department of Orthopedic Surgery, Yonsei University Health System, Seoul, Republic of Korea

⁴Department of Internal Medicine IV, Nephrology and Hypertension, Medical University of Innsbruck, Innsbruck, Austria

⁵Department of Health, Medicine and Caring Sciences, Linköping University, Linköping, Sweden

⁶Department of Pediatrics, Yonsei University College of Medicine, Seoul, Republic of Korea

⁷Severance Underwood Meta-Research Center, Institute of Convergence Science, Yonsei University, Seoul, Republic of Korea

Correspondence to Jae Il Shin Department of Pediatrics, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul 03722, Republic of Korea E-mail: shinji@yuhs.ac

*Seungyeon Kang and Se Jin Park contributed equally to this work as the co-first authors.

Received 2025 January 24; Revised 2025 February 19; Accepted 2025 February 20.

Abstract

Genetic kidney diseases are caused by mutations in specific genes that significantly affect kidney development and function. Although the underlying pathogenic genes of many kidney diseases have been identified, an understanding of their mechanisms and effective treatments remains limited. Gene editing, particularly using clustered regularly interspaced short palindromic repeats (CRISPR), has recently become a promising approach for studying genetic diseases and the CRISPR/CRISPR-associated protein 9 (CRISPR-Cas9) method has become a prominent research method. It has been shown that CRISPR-Cas9 can be targeted to knock out specific genomic sites, which enables researchers to correct gene mutations, prevent inheritance, and better understand the function of genes and the effectiveness of drugs. However, the application of CRISPR-Cas9 technology in the development of therapeutic agents against genetic kidney disease has been overlooked compared with other genetic diseases. In this paper, we provide an overview of the current research advancements in genetic kidney diseases using CRISPR technology, as well as the diverse preclinical research methods implemented, with particular emphasis on autosomal dominant polycystic kidney disease.

Keywords: Autosomal dominant polycystic kidney disease; Clustered regularly interspaced short palindromic repeats; CRISPR-associated protein 9; Gene editing; Kidney

Introduction

Genetic kidney diseases (GKDs) are a group of rare diseases with a prevalence of approximately 60 to 80 per 100,000 individuals [1]. GKD is one of the leading causes of early-onset chronic kidney disease, affecting 10% of adults globally [2,3]. It ranks fifth among the causes of end-stage kidney disease [4]. Furthermore, it is responsible for approximately 10% to 15% of adult kidney replacement therapy cases [5].

Most GKDs are monogenic, which means that the disease is caused by a single gene mutation [6]. This characteristic of GKDs enables the use of site-specific nuclease systems to study the genetic characteristics of the disease. In addition, as mutated gene expression occurs in vivo, gene editing technology is an effective method to control the development of diseases at the genetic level [7]. Clustered regularly interspaced short palindromic repeats (CRISPR) is a site-specific nuclease gene editing system that can permanently correct destructive base mutations and disease-causing genes, and it has emerged as a new technology for future genetic disease treatment and research. The potential of CRISPR as a genome-editing tool was first recognized in 2012 and was further conventionalized in 2013 [8-11]. Applications of CRISPR technology are of considerable interest because of their diverse possibilities as therapeutic approaches for genetic diseases [12,13]. The ability of CRISPR technology to edit gene mutations and generate in vivo and in vitro models that can reflect genetic abnormalities has expanded our knowledge of the mechanisms and pathogenesis of genetic diseases.

CRISPR is a powerful tool for understanding the pathogenesis of diseases, the pathological characteristics of cells and tissues, and the constructive environment of diseases. Insights gained from these characteristics will enrich the development of gene-targeted therapeutic drugs and offer further knowledge of genetic disease treatments. This review provides a comprehensive understanding of the topic by examining current advances in our knowledge of the genetic basis of autosomal dominant polycystic kidney disease (ADPKD) and potential therapeutic strategies using CRISPR.

The CRISPR system

CRISPR is part of an adaptive immune system that protects against foreign invasive materials in bacterial and archaeal genomes [14]. In 2012, two studies discovered the potential of CRISPR as a genetic editing tool by programming it with RNA to target specific genomic sites [8,9]. In 2013, several studies further demonstrated the ability of CRISPR as a genetic engineering tool using small guide RNAs (sgRNAs) [9-11].

The CRISPR/CRISPR-associated protein 9 (CRISPR-Cas9) system comprises a Cas9 protein and a sgRNA. Typically, sgRNAs are 100 nucleotides long and the Cas9 protein binds to the last 80 nucleotides to form a ternary complex [10,11,15]. The remaining 20 nucleotides are in a free state, and thus, they bind to eukaryotic DNA based on their complementary sequences [9,16]. Through this mechanism, the first 20 nucleotides of the sgRNA guide the Cas9 protein to a specific target site in the DNA. By manipulating the first 20 nucleotide sequences, the CRISPR system can be programmed to target different genomic sites. After binding to the genome, the DNA-cutting enzyme cuts DNA at the third and fourth nucleotides upstream of the protospacer adjacent motif, introducing a double-stranded break (DSB) at the target site [8,9]. These DNA DSBs need to be rapidly repaired because they are fatal to the genome. This is accomplished through an endogenous DSB repair system involving non-homologous end joining (NHEJ) and homology-directed repair (HDR) [8,9]. NHEJ trims and ligates DNA, causing insertion and deletion mutations that can interfere with the open reading frames [8,9]. HDR repairs DNA based on a template strand within the genome or supplied in an experiment. Although HDR is more precise and accurate, the primary repair method is NHEJ because HDR is inefficient for practical use [15,10,17].

Due to its simplicity in targeting the genome by altering the sgRNA nucleotide sequence, CRISPR-Cas9 has emerged as a convenient and cost-effective platform. Current research on its therapeutic applications in the treatment of genetic diseases has demonstrated its wide applicability to diverse disease phenotypes.

ADPKD and CRISPR

ADPKD is one of the most common genetic conditions, affecting an estimated 12.5 million people globally [18]. In ADPKD, the microscopic tubules progressively expand into fluid-filled cysts that replace the normal parenchyma [19]. Furthermore, the cysts can cause bilateral kidney enlargement and renal failure. ADPKD is inherited via heterozygous loss-of-function mutations in PKD1 and PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively, and in rare circumstances, it is caused by other mutations [20,21]. Mutations in PKD1 and PKD2 account for approximately 78% and 13% of ADPKD cases, respectively [22]. It has been hypothesized that cyst initiation occurs from germline inactivation mutations in one allele and somatic inactivation of PKD1 or PKD2 [23,24]. However, recently, a gene dosage (haploinsufficiency) model hypothesized that complete loss of the PKD gene is unnecessary and that renal cyst formation occurs when the PKD1 dosage falls below a critical threshold [25,26].

Applications using stem cells

The potential of the use of stem cells in research is widely understood because of their ability to self-renew and differentiate into various somatic cell types and tissues under different culture conditions [27-30]. Somatic cells from human patients are conventionally converted into induced pluripotent stem cells (iPSCs), which are then subjected to patient-specific or gene-targeted mutations. The most common protocol for introducing the CRISPR-Cas9 system into iPSCs involves the use of viral vectors to deliver the guide RNA and Cas9 protein. CRISPR-Cas9 technology has been applied to stem cells for research on GKDs [31].

Many studies have implemented CRISPR in stem cells to accurately recapitulate kidney organoids and genetically model ADPKD for future research. Using human pluripotent stem cell (hPSC)-derived kidney cells, Freedman et al. [32] found that loss-of-function PKD mutations resulted in cyst formation, using CRISPR to introduce PKD1 and PKD2 mutations in hPSC-derived kidney cells. They also found that PKD-specific cystogenesis in the tubules is a cell-intrinsic phenomenon; thus, human kidney disease can be recapitulated in vitro. Their study provided a three-dimensional (3D) culture system that establishes innovative cellular systems for studying human renal physiology and pathophysiology in vitro. Cruz et al. [33] established a genetic model of PKD cystogenesis that implicates the microenvironment in the early stages of ADPKD. They developed the PKD model by introducing biallelic mutations in PC1 and PC2 to hPSC, using CRISPR. The successful removal of adherent cues increased cystogenesis 10-fold, leading to the production of cysts resembling PKD, which expand to a diameter of 1 cm. Defects in PC1 expression and collagen compaction were observed after stromal removal, which enabled the outgrowth of PKD cell lines. These findings provide critical insights into the modeling of PKD using human cellular systems. Furthermore, it was found that cyclic adenosine monophosphate (cAMP) induces cysts in both PKD and non-PKD kidneys. This suggests that strengthening stromal or scaffolding components may provide a cue favoring migratory repair and that PC1 may function as an adhesion regulator to maintain tubular architecture through its interactions with the microenvironment. Huang et al. [34] demonstrated that the inhibition of p38 mitogen-activated protein kinase activity leads to long-term expansion of mouse and human nephron progenitor cells (NPCs) in vitro in a two-dimensional (2D) culture setting, proposing a new rapid, efficient, and scalable organoid model of PKD. This finding was made by identifying experimental observations from PKD2 gene-knocked human NPCs, using CRISPR. The activation of yes-associated protein signaling programs in hPSC-derived induced NPCs to the primary state of NPCs allows enhanced modeling of diseases that affect the distal nephron of the kidney, including ADPKDs. In addition, PTC-209 and other B-cell-specific Moloney leukemia virus insertion site 1 inhibitors have been shown to be potential candidates for future PKD treatments.

Further research into potential therapeutics is possible using stem cells and data from previous studies on the generation of ADPKD kidney organoids. Vishy et al. [35] reported eukaryotic ribosomal-selective glycosides (ERSGs) as potential PKD therapeutics that enable ribosomal readthrough of nonsense mutations. CRISPR has been used to introduce four common nonsense mutations into iPSCs, which were then differentiated into kidney organoids and treated with ERSGs. ERSGs may prevent cyst initiation and limit the growth of preformed cysts by partially restoring PC expression.

Many studies on the use of pluripotent stem cells have focused on the generation of kidney organoids (nephron organoids) [36]. Cyst formation in ADPKD involves the formation of nephron progenitors and ureteric buds [37]. Large renal cysts originate from ureteric buds rather than from nephron progenitors. Thus, modeling cyst formation in ureteric bud organoids is critical for the future treatment of ADPKD. Kuraoka et al. [38] evaluated cyst formation in nephron progenitors and ureteric buds by developing nephron progenitor and ureteric bud organoids with PKD1 gene mutations for iPSCs using CRISPR. Upon stimulation with cAMP, enhanced cystogenesis was observed in organoids lacking PKD1 or PKD2. Moreover, consistent with previous studies, it has been shown that cAMP signaling is required for in vitro cystogenesis in both nephron and ureteric bud organoids. After observing the expression of arginine vasopressin receptors, only ureteric organoids responded to vasopressin to form cysts. This provides a valuable basis for successful ADPKD modeling and drug screening. Observations of cyst formation in organoids with heterozygous PKD1 mutations suggest that epithelia may impact cyst formation, with contributions toward cystogenesis via exogenous signals.

Applications using cells

CRISPR-Cas9 can be directly used in specific renal cells. Establishing cellular models of disease has long been effective at testing the hypotheses of disease pathogenesis. In addition, they may serve as potential targets for disease treatment. Huang et al. [39] modeled ADPKD using SIX2⁺ NPCs to identify small-molecule inhibitors. They differentiated induced NPCs and human NPCs in 2D and 3D culture formats using human neuropeptide S receptor-v2 medium to generate nephron organoids. CRISPR was then applied to introduce Pkd1^−/− and Pkd2^−/− mutations to generate cystic nephron organoids. Incubation of the organoids with PTC-209 resulted in a dose-dependent cyst-inhibitory effect without cellular toxicity. Pkd2^−/− mutated organoids showed co-expression of lotus tetragonolobus lectin and cadherin 1 in the cyst-lining cells, increased cellular proliferation, enhanced expression levels of cell-cycle-related genes, and increased activity of the mammalian target of rapamycin pathway and MYC7. Additionally, the oxygen consumption and extracellular acidification rates were elevated. Treatment of organoids with cystic fibrosis transmembrane conductance regulator inhibitor 172, metformin, AZ505, and tubacin inhibited cyst formation, which is consistent with the results of previous studies [40-44]. Chumley et al. [45] evaluated extracellular acidification and glucose metabolism in human embryonic kidney cell lines using PKD2. Their findings revealed that mutations in PKD2 (introduced by CRISPR) increased the overall extracellular acidification over time and altered mitochondrial morphology to resemble PC1 deficiency. These findings suggest the potential application of targeted energy metabolism as a therapeutic approach for PKDs. Another approach used by Porath et al. [46] showed that mutations in the GANA gene cause ADPKD and defects in PC1 maturation, resulting in cystogenesis. After knocking out the GANAβ gene using CRISPR-Cas9, loss or reduction of GIIα was observed, leading to PC1 and PC2 maturation and localization defects that caused ADPKD.

Applications using animal studies

Despite ongoing debates regarding the ethical considerations of genome editing in animals, it is an effective method for understanding the pathology of human diseases [47,48]. Moreover, it can be used to elucidate the mechanisms underlying disease progression and development [49,50]. Watanabe et al. [51] induced a PKD phenotype by generating PKD1-heterozygous-knockout (PKD1^insG/+) pigs using CRISPR-Cas9 and somatic cell cloning techniques. They found that PKD1^insG/+ pigs developed many pathological conditions similar to those of patients with heterozygous mutations in PKD, with pathological similarities in the formation of macroscopic kidney cysts at the neonatal stage, the number and cystogenic dynamics of the kidney cysts formed, interstitial tissue fibrosis, and the presence of a premature asymptomatic stage. These findings demonstrate the potential of PKD1^insG/+ pigs to be used to study early interventions in pediatric patients with ADPKD, to verify the effects of prophylactic treatment, and to test long-term treatment. Further research using animal models will reveal new insights into the disease and future treatment methods. Tsukiyama et al. [52] generated cynomolgus monkey models with ADPKD PKD1 mutations to demonstrate how animals recapitulate the key pathological features of human diseases. They used CRISPR-Cas9 to generate PKD1-knockout or mosaic monkeys with different degrees of cyst formation. Their research identified the lineage of identities of cyst epithelia and found that most cysts in heterozygotes were derived from the distal tubules, which reflects the initial stages of cystogenesis. They also found that the formation of cysts in the collecting ducts was significantly associated with the severity of the cysts. These results suggest that distal tubules may be a potential drug target for patients with PKD1. Soomro et al. [53] reported that discoidin domain receptor 1 (DDR1) is not a viable drug target for ADPKD after in vivo genetic deletion of DDR1 using CRISPR-Cas9 in a mouse model. Mice with ADPKD showed no decrease in cyst growth or preservation of kidney function, suggesting that the pathogenesis of PKD does not involve DDR1 expression. Table 1 shows the previous findings of ADPKD models developed using CRISPR-Cas9 technology.

Table 1.

Summary of findings in the previous studies regarding ADPKD using CRISPR-Cas9 systems

Conclusions

CRISPR-Cas9 technology is a new, promising technique that opens new frontiers for diverse research into the genetics of diseases. The recent introduction of this technology has greatly increased the number of studies on GKD. Methods utilizing stem cells, podocytes (cells), and animals have been used in research on GKDs involving CRISPR-Cas9. These studies have aimed to construct an accurate disease model that demonstrates human disease progression and development and to understand the mechanism of diseases to identify possible treatment methods. This comprehensive review focuses on the application of CRISPR technology in research on GKDs, especially ADPKD, and explains the methods used in detail. It summarizes robust preclinical data and identifies effective approaches that have not been considered in studies using other gene editing technologies. Moreover, it provides an updated overview of the research conducted by other scientists on GKDs. Although studies on using CRISPR for in vivo gene therapy for ADPKD are yet to be continued, emerging approaches for investigating the genetic and phenotypic characteristics of diseases using CRISPR may be promising for future research on new potential treatments for inherited diseases.

Notes

Conflicts of interest

Jae Il Shin is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflicts of interest relevant to this article were reported.

Funding

This work was supported by the Yonsei Fellowship, funded by Lee Youn Jae (JIS). This study was performed as the research internship program of Yonsei International Summer School (SK).

Author contributions

Conceptualization: SK, SJP, JIS

Data curations: SK, SJP, MHL, AK, JIS

Formal analysis: SK, MHL

Investigation: SK, MHL

Methodology: SK, SJP, JIS

Project administration: SJP, JIS

Visualization: SK, SJP

Writing-original draft: SK, SJP

Writing-review & editing: SK, SJP, MHL, AK, JIS

All authors read and approved the final manuscript.

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Table 1.

Summary of findings in the previous studies regarding ADPKD using CRISPR-Cas9 systems

Author (year)	Disease	Method	Target mutation area	Study findings
Vishy et al. (2024) [35]	ADPKD	Human pluripotent stem cell (kidney organoids)	PKD1-R2430X, PKD1-Q3838X, PKD2-R186X, PKD2-R872X, PKD1-R2430X, and PKD2-R872X	- Eukaryotic ribosomal-selective glycosides: cyst initiation and growth of preformed cysts ↓ by restoring polycystin expression.
				- Aminoglycoside drugs: cysts ↓ via ribosomal readthrough.
				- Fluorescent aminoglycosides: accumulate in kidneys and PKD cysts in mice in vivo.
				- Heterozygosity: cyst formation ↓ and base-editing gene therapy.
Freedman et al. (2015) [32]	ADPKD	Human pluripotent stem cell (kidney organoids)	PKD1 and PKD2	- Loss-of-function PKD mutations → cyst formation.
Freedman et al. (2015) [32]	ADPKD	Human pluripotent stem cell (kidney organoids)	PKD1 and PKD2	- PKD-specific cystogenesis from tubules: cell-intrinsic phenomenon.
Huang et al. (2023) [34]	ADPKD	Human-induced pluripotent stem cell	p38 and YAP	- Inhibition of p38 MARK activity → long-term expansion of mouse and human NPCs in vitro in the 2D culture setting.
Huang et al. (2023) [34]	ADPKD	Human-induced pluripotent stem cell	p38 and YAP	- PTC-209 or other BMI-1 inhibitors: prospective candidates for PKD treatment.
Cruz et al. (2017) [33]	ADPKD	Induced pluripotent stem cell (kidney organoids)	PKD1 and PKD2	- Removal of adherent cues: cystogenesis ↑.
Cruz et al. (2017) [33]	ADPKD	Induced pluripotent stem cell (kidney organoids)	PKD1 and PKD2	- Removal of stroma → outgrowth of PKD cell lines.
Kuraoka et al. (2020) [38]	ADPKD	Induced pluripotent stem cell (UB organoids)	PKD1	- Both nephron and UB organoids: cyst formation upon forskolin treatment.
Kuraoka et al. (2020) [38]	ADPKD	Induced pluripotent stem cell (UB organoids)	PKD1	- UB organoids (not nephron organoids): responding to vasopressin to form cysts.
Chumley et al. (2019) [45]	ADPKD	Cell (HEK-293)	PKD1 and PKD2	- Mutations in PKD genes → changing mitochondrial energy metabolism.
				- Mutations in PKD1 and PKD2 : overall extracellular acidification ↑.
				- PKD1 mutations: non-glycolytic acidification rates and tricarboxylic acid cycle activity ↑ or breakdown intracellular glycogen, and basal and ATP-linked oxygen consumption rates ↑.
				- PKHD1 and PKD2 mutations: altering mitochondrial morphology (resembling PC1 deficiency).
Porath et al. (2016) [46]	ADPKD	Cell (RCTE)	GANAβ	- Knockout of GANAβ → loss or reduction of GIIα, and PC1 and PC2 maturation, and localization defects that cause ADPKD.
Huang et al. (2024) [39]	ADPKD	Cell (NPCs)	PKD1 and PKD2	- Cystic organoids: responding to CFTRinh172, metformin, AZ505, and tubacin, but not tolvaptan.
Huang et al. (2024) [39]	ADPKD	Cell (NPCs)	PKD1 and PKD2	- Incubation of organoid with PTC-209 → dose-dependent cyst inhibitory effect without cellular toxicity.
Tsukiyama et al. (2019) [52]	ADPKD	Monkey model	PKD1	- Cyst formation in the collecting ducts: associated with cyst severity.
Tsukiyama et al. (2019) [52]	ADPKD	Monkey model	PKD1	- Abnormal function of PKD1: relate to high rate of abortion.
Watanabe et al. (2022) [51]	ADPKD	Pig model	PKD1	- Heterozygous PKD1 pigs → many pathological conditions similar to ADPKD patients.
Soomro et al. (2019) [53]	ADPKD	Mouse model	DDR1	- DDR1: no playing a role in PKD pathogenesis.
Soomro et al. (2019) [53]	ADPKD	Mouse model	DDR1	- DDR1: not a viable drug target for ADPKD.

CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR-associated protein 9; ADPKD, autosomal dominant polycystic kidney disease; PKD, polycystic kidney disease; MARKs, mitogen-activated protein kinases; NPCs, nephron progenitor cells; 2D, two dimensional; BMI-1, B-cell-specific Moloney leukemia virus insertion site 1; UB, ureteric bud; HEK-293, human embryonic kidney 293; PC1, polycystin-1; RCTE, renal cortical tubular epithelial; GIIα, glycoprotein α-subunit of glucosidase II; PC2, polycystin-2; CFTRinh172, cystic fibrosis transmembrane conductance regulator inhibitor 172; DDR1, discoidin domain receptor tyrosine kinase 1.