Progressive, late-onset, nonsyndromic sensorineural hearing loss is the most prevalent sensory disorder in humans and encompasses various clinical entities such as presbyacusis, noise-induced hearing loss, drug ototoxicity, and most autosomal dominant types of hearing loss. Presbyacusis or age-related hearing loss is a widespread cause of sensory impairment globally and thus represents a significant health and socioeconomic burden for all populations
[1-2]. The purinergic receptor P2X2 (
P2RX2) gene serves as an ion channel the receptor for adenosine triphosphate (ATP) with permeability to cations including Na
+, K
+, and Ca
2+ (with Ca
2+ being the most permeable)
[3]. Loss of P2RX2 function has been identified as the underlying cause of autosomal dominant nonsyndromic deafness 41 (DFNA41), which renders patients more susceptible to noise-induced damage while also experiencing high-frequency hearing loss and accelerated presbyacusis.
The zebrafish (
Danio rerio), commonly referred to as “zebra fish”, is a teleost fish. Indigenous to India and Bangladesh, it is a tropical freshwater fish that exhibits gentle temperament and has an omnivorous diet
[4-5]. In comparison with other animal models, the zebrafish possesses advantageous characteristics such as its small size, ease of maintenance, rapid development rate, robust reproductive capacity, ability to reach sexual maturity at 3 months old, capability for in vitro fertilization and embryo development in vitro. Additionally, its transparent nature facilitates easy accessibility and observation. Furthermore, the high degree of genetic similarity between zebrafish and humans (up to 87% identity) positions this species as an ideal model organism for investigating human diseases and developmental mechanisms
[6-7]. Compounds identified through screening using zebrafish have been used or will soon be employed in clinical settings. Consequently, zebrafish is gaining recognition as a valuable therapeutic guide for patients afflicted with cancer or rare diseases
[8-9]. The
p2rx2 gene resides on chromosome 5 of the zebrafish genome and consists of 12 exons along with 11 introns. Its full-length complementary DNA (cDNA) sequence spans 1 292 bp encoding a protein comprising 400 amino acids. Notably,
p2rx2 encompasses 4 evolutionarily conserved functional domains that influence extracellular ATP-gated cation channel activity and ion migration
[10]. Moreover, by employing differential gene expression profiling combined with genomic association analysis techniques during early stages of human embryonic development revealed widespread expression of
p2rx2 across multiple tissues; particularly notable was its presence within the otic vesicle
[11].
The normal function of P2RX2 in the inner ear is crucial for auditory perception, making it a significant area of research and potential avenue for gene therapy in treating progressive deafness and noise-induced hearing loss
[12]. To gain deeper insights into
p2rx2 gene variants associated with deafness, we employed CRISPR/Cas9 gene editing system to establish a
p2rx2 knockout zebrafish model, facilitating our understanding of the molecular mechanisms underlying
p2rx2-related hearing loss and enabling the development of novel therapeutic strategies.
1 Materials and methods
1.1 Ethics statements
This study was approved by the Ethics Committee of the Second Xiangya Hospital, Central South University (Approval number: 20250021).
1.2 Experimental animals
The Tübingen (TU) strain zebrafish utilized in the experiments described in this study was derived from the State Key Laboratory of Developmental Biology of Freshwater Fish of Changsha culture. The water temperature was maintained at 28 ℃, with the pH ranging from 6.5 to 7.5, and subjected to alternating light (14 hours) and dark (10 hours) cycles. Embryos were cultured in E3 medium at a constant temperature of 28.5 ℃ until their membranes ruptured after 2 days post-fertilization (dpf). From day 5 onwards, the embryos were fed primarily with Paramecium for approximately 2 weeks, after which their diet was transitioned to harvested worms from the culture systems on the shelf. Microinjection procedures were generally conducted during the zebrafish embryo’s one-cell stage development.
1.3 Experimental reagents
The primers, DNA marker, and polymerase chain reaction (PCR) high-fidelity enzyme were synthesized or purchased from Engine Biology Co., Ltd. (China). Agarose, bis-acrylamide, and ammoniumpersulfate (APS) were purchased from BBI Life Sciences Co., Ltd. (China). Acrylamide were purchased from Sangon Biotech Co., Ltd. (China). Tris base were purchased from Phygene Company (China). Hydrochloric acid were purchased from Sinopuncture Group Chemical Reagent Co., Ltd. (China). TEMED were purchased from Beijing Prialai Gene Technology Co., Ltd. (China). The PCR product purification kit was acquired from Sangon Bio Co., Ltd. (China), while the in vitro transcription kit and Cas9 protein were obtained from Thermo Fisher Scientific Inc. (USA). The RNA purification kit used in this study was purchased from Qiagen (Germany).
1.4 Design of single-guide RNA target sites
The complete sequences of target genes, including the transcript and relevant information, were obtained from the Ensembl website. All candidate target sequences adjacent to 5'-NGG-3' (PAM) were identified, with the typical size ranging from 18 to 20 bp for the target sequences. The forward primer for the single-guide RNA (sgRNA) was designed by adding a protective base (5'-GCG-3') and either T7 promoter (5'-TAATACGACTCACTATA-3') or Sp6 promoter (5'-ATTTAGGTGACACTATA-3') before the target sequence. Additionally, the upstream sequence of sgRNA backbone sequence (5'-GTTTTAGAGGCTAG-AAATAGG-3') was added after the target sequence. Based on the target site of the
p2rx2 gene, the forward (F) and reverse (R) genomic detection primers,
p2rx2-F and
p2rx2-R, were designed using Primer 3.0 (
Table 1).
Based on the aforementioned method, gene knockout sites were identified and selected within the
p2rx2 gene sequence. Protective bases and promoter sequences were incorporated upstream of the target site sequence, while upstream sequences of sgRNA backbone were added downstream of the target site sequence, serving as forward primers
sgRNA1-F and
sgRNA2-F respectively. The reverse primer
sgRNA-R was designed based on the sequence downstream of the sgRNA skeleton. The selection of the
p2rx2 gene target site and the subsequent gene knockout procedure are illustrated in
Figure 1.
1.5 Synthesis of sgRNA
The sgRNA template sequences of the p2rx2 gene with a T7 promoter were obtained by PCR amplification using sgRNA1-F/sgRNA-R or sgRNA2-F/sgRNA-R as primers, employing a high-fidelity enzyme at an annealing temperature of 62 ℃ and an extension time of 10 seconds. Subsequently, the PCR products were analyzed through agarose gel electrophoresis and gel cutting recovery. The recovered PCR products served as templates for synthesizing sgRNA using the T7 in vitro transcription kit. The resulting transcript products were purified and recovered utilizing an RNA purification kit, followed by agarose gel electrophoresis and concentration measurement prior to storage at -80 ℃.
1.6 Microinjection and assessment of target site efficacy
The fertilized zebrafish eggs were collected and arranged on the injection plate within 15 minutes of reaching the 1-cell stage. The p2rx2 gene target sgRNA1 (103.26 ng/μL), sgRNA2 (110.26 ng/μL), and Cas9 protein (150 to 300 ng/μL) were mixed in a ratio of 1꞉1꞉0.7, respectively. Approximately 1 nL of the mixed solution was co-injected into the zebrafish fertilized eggs using a quantitative microinjection system, followed by incubation at 28.5 ℃.
To validate the efficacy of the target site, we cultured the injected embryos until 36 hours post-fertilization (hpf). After cultured for 36 hours, some wild type embryos and some injected embryos were collected for injection efficiency identification, while the remaining embryos were cultured until adulthood.
1.7 Screening of genetically stable mutants
The injected embryos were cultured to develop into juveniles at about 2 months of age, and the juveniles were genotyped by tail clipping one by one. Because the distance between the 2 target sites is 47 bp, when the 2 target sites are effective, a large fragment of deletion will be caused, and then the genome will be repaired. Because there is no template to guide the DNA repair is imperfect, which will lead to large fragment deletion or large fragment insertion of the gene. The genomes were amplified by PCR using genomic detection primers, followed by agarose gel electrophoresis or polyacrylamide gel electrophoresis (PAGE). If the PCR products showed both the wild type target band and a smaller band (47 bp) than the wild type target band, these larvae were considered F0 (first generation 0) fish carrying the mutation. Juveniles of the F0 were continued to be raised for about one month until they became adults and crossed with the wild type. Fish that could be stably inherited were selected by the same genotype identification method, and these fish were filial generation 1 (F1) mutants. After genotype identification, according to the results of agarose gel electrophoresis, the band smaller than the target band of the wild type in the F1 mutant was cut and recovered, and sent to the Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The mutation site and the number of mutated bases were analyzed according to the sequencing results.
1.8 Staining treatment of <italic>p2rx2</italic> hair cells
The formation of hair cells in the zebrafish head was assessed by YO-PRO-1 (a fluorescent nucleic acid stain) staining to investigate the impact of p2rx2 knockout. Initially, both p2rx2 filial generation 2 (F2) mutant embryos and wild type embryos were collected. A mixture of YO-PRO-1 dye and E3 water at a ratio of 1꞉1 500 was added to an 8-well plate at 2.5 dpf, followed by labeling and placement of 50 wild type and p2rx2 gene mutant embryos per well. Subsequently, fluorescence microscopy was employed to observe and capture images of the expression of hair cells in p2rx2 gene mutant zebrafish at 3, 5, and 7 dpf.
1.9 Auditory evoked potential
A total of fifteen 4-month-old zebrafish, including wild type and
p2rx2 mutant zebrafish, were allocated into 2 groups for auditory evoked potential (AEP) detection. The AEP measurement device was set up according to a previously described protocol
[13], using Tucker-Davis Technology (TDT, Alachua, FL, USA) hardware and software for acoustic stimulation and AEP signal recording. The experiment was conducted on an anti-vibration air flotation table inside a soundproof room. A rectangular water tank (50 cm×35 cm×23 cm), fitted with built-in bottom underwater speakers (UW30) and a sand layer, was used as the experimental chamber. Low-frequency sound stimuli (<300 Hz) were generated by driving an acrylic vibration plate via an external mini vibrator (Bruel & Kjær 4810), which was connected to the side wall of the tank through a waterproof stainless steel rod positioned 7 cm from the fish’s head. Adult zebrafish were mildly anesthetized with MS-222 (120 mg/L), then immobilized in a custom-made sponge frame that left only their heads exposed, and placed 8 cm underwater, aligned with the center of the speaker. Spontaneous breathing resumed during the recording session. Water temperature was maintained at (25±1) ℃ using a circulating zebrafish housing system. During recording, 2 insulated stainless-steel electrodes were placed under microscopic guidance—one on the skin overlying the brainstem (recording electrode) and the other on the lateral body surface (reference electrode). The signal was amplified using a preamplifier (RA4PA, 20× gain), bandpass-filtered between 0.1 and 1 kHz, and then digitized by a TDT system (RZ6 processor) with 16-bit resolution and ±4 mV input range. Stimulus generation and AEP acquisition were controlled using BioSig software. Testing and recording were performed using TDT RZ6 instrument. Given that the optimal hearing range for zebrafish is between 600 and 1 000 Hz
[9], tone bursts at frequencies of 600, 800, 1 000, and 2 000 Hz with a duration of 20 ms and rise/fall time of 2 ms (Blackman window gating) were employed. The acoustic stimulus intensity was reduced from 82 to 150 dB in decrements of 4 dB per step. The first single peak with the highest sound level was identified as an auditory signal while the lowest sound level at which this signal could be detected was defined as the threshold.
1.10 Statistical analysis
Statistical analysis was performed using GraphPad 9 software. Data were presented as means±standard deviation. The AEP thresholds for individual frequencies were assessed using a two-way analysis of variance (ANOVA) in combination with Sidak’s multiple comparison test. P<0.05 was considered statistically significant.
2 Results
2.1 Efficacy and analysis of<italic> p2rx2 </italic>gene injection
The injected embryos were cultured until 36 hpf, and 13 tubes containing 2 embryos each were randomly selected, and genomic DNA was extracted for PCR amplification (
Figure 2). The results revealed an additional band above lane 6, alongside a wild type band of size 278 bp, providing evidence for successful insertion of the sequence.
2.2 Screening of F0 embryo juvenile
The successfully injected embryos were raised to adulthood. PCR detection of genomic DNA from these adults revealed an additional higher molecular weight band above lanes 1 and 3, alongside the presence of a wild type band measuring 278 bp (
Figure 3).
2.3 Screening of stable genetic mutants
The first F0 mutant was outcrossed with wild type zebrafish, consistent detection of the mutant allele through PCR-based genotyping of F1 embryos confirmed successful inheritance of the mutation (
Figure 4A). The 344 bp band was excised and sent for sequencing, revealing an insertion before the first target site (
Figure 4B). Comparison with the National Center for Biotechnology Information (NCBI) blast database confirmed a 66 bp insertion before the first target site of the mutant, demonstrating effective targeting resulting in large fragment insertion (
Figure 4C). Stable germline transmission was verified in adult F1 zebrafish through PCR-based genotyping (
Figure 4D). Stable inheritance of the F1 fish was achieved. Subsequent sequencing analysis revealed that although a frameshift mutation did not occur due to a 66-base insertion, bases at position 670 to 672 were TAA introducing a stop codon leading to premature termination of gene translation and consequent loss of protein function.
2.4 Phenotype of <italic>p2rx2 </italic>gene homozygous mutants
In this experiment, we performed intercrossing of the F1 of
p2rx2 gene heterozygous mutant adult fish and observed the phenotype of F2 of
p2rx2 gene homozygous mutant embryos. The results revealed normal appearance and no observable phenotype for the
p2rx2 gene (
Figure 5A). Subsequently, we conducted further verification on embryos exhibiting normal development, confirming their status as homozygous mutants with gene insertion (
Figure 5B).
2.5 Changes in auditory hair cells
In this study, hair cells in F2
p2rx2 homozygous mutant zebrafish were stained and imaged. The results demonstrated that at 5 dpf, no significant differences were observed in the number or arrangement of hair cells within the lateral line region between
p2rx2 gene knockout zebrafish and the control group (
Figure 6). In contrast, a marked reduction in hair cell density was evident in the otolith region of
p2rx2 gene knockout zebrafish compared to controls (
Figure 7).
2.6 AEP test results
The AEP thresholds of the homozygous mutant zebrafish with the
p2rx2 gene were significantly higher compared to those of the wild type zebrafish at stimulation frequencies of 600, 800, 1 000, and 2 000 Hz (all
P<0.001,
Figure 8).
3 Discussion
CRISPR/Cas9 gene editing system has gained significant popularity in recent years as an efficient tool for targeted gene knockout
[14-15]. In this study, we employed the zebrafish model organism to knock out the
p2rx2 gene using CRISPR/Cas9 gene editing system. Leveraging the advantages of zebrafish as a model organism and the simplicity of CRISPR/Cas9 system gene editing technology, we utilized cloning-free PCR with oligonucleotides as templates. Following purification and recovery, the PCR products were directly used for sgRNA synthesis, streamlining the process of gene knockout while significantly enhancing experimental efficiency and minimizing potential risks
[16-18].
The CRISPR/Cas9 gene editing technology carries a relatively high risk of off-target effects; therefore, this study imposed more stringent criteria for the rational selection of target sites. To generate a p2rx2 gene loss-of-function mutant,2 target sites located 66 bp apart were designed within the gene sequence, and the corresponding 2 sgRNAs were co-injected into one-cell stage zebrafish embryos. If both sites were efficiently cleaved, large-scale deletions or insertions could occur in the genomic region between them, leading to a reduction in the number of encoded amino acids and consequently disrupting the normal structure and function of the protein. These large-scale mutations can be readily detected by agarose gel electrophoresis without the need to assess each individual target site. In this experiment, we successfully obtained a 66-nucleotide insertion mutation; however, due to the introduction of TAA within this insertion, premature translation termination occurred resulting in the generation of a loss-of-function strain.
Due to the mosaic nature and potential germline transmission failure in F0 mutants, large-fragment insertion carriers were identified and outcrossed with wild-type zebrafish to establish the F1 generation.The mutation in F1 mutants was stably inherited. Upon intercrossing of the heterozygous F1 mutants, 25% of the offspring displayed homozygosity, aligning with the expected outcomes. The resulting homozygous mutant zebrafish in the F2, derived from intercrossing of the heterozygous F1 adult fish, developed normally without discernible phenotypic abnormalities. This suggests that gene insertion did not impede overall embryonic development and did not induce any noticeable defects in zebrafish embryos subsequent to p2rx2 mutation. Further investigations revealed that p2rx2 knockout led to aberrant hair cell development within the otolith, which serves as a hearing structure in zebrafish.
In contrast to mammals, zebrafish exhibit the typical inner ear anatomy of vertebrates, albeit lacking an outer ear and middle ear. While the zebrafish inner ear does not possess a dedicated auditory organ akin to the mammalian cochlea, it does have otolith organs resembling the mammalian vestibule. The saccule in zebrafish is responsible for auditory perception, while the utricle, semicircular canals, and lymphatic vessels primarily serve vestibular functions. It is noteworthy that larval zebrafish can perceive sound frequencies ranging from 100 to 4 000 Hz similar to adult zebrafish at 5 dpf. Additionally, zebrafish possess another sensory nerve organ called the lateral line, which detects local water flow and low-frequency vibrations while regulating behaviors such as learning, escape responses, and feeding
[19]. The lateral line system on the body surface of zebrafish consists of numerous individual neuromasts with centrally innervated clusters of sensory hair cells. Lateral line hair cells and inner ear hair cells share functional and molecular similarities
[20]. However, in our
p2rx2 gene knockout model, otoconia hair cells exhibited evident developmental and functional abnormalities whereas lateral line hair cells developed normally. Further investigation is required to elucidate the potential molecular mechanisms involved. Our future studies will employ
p2rx2 knockout models to explore these mechanisms underlying hair cell development and hearing regulation.
The p2rx2 knockout zebrafish model was successfully established in this study, providing a solid foundation for subsequent functional investigations of the p2rx2 gene.
the Natural Science Foundation of Hunan Province(2023JJ30753)
the Innovative Construction Foundation of Hunan Province(2023SK4030)
the Natural Science Foundation of Changsha(kq2208326)
,
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