PKHD1L1 gene point mutation rat model, construction method therefor, and detection method therefor

Abstract

The present invention belongs to the technical field of animal modeling. Particularly disclosed are a PKHD1L1 gene point mutation rat model, a construction method therefor, and a detection method therefor. In the present invention, a murine P.L867S is knocked into the PKHD1L1 gene on the basis of a CRISPR / Cas9 system, and a point mutation is performed to change corresponding bases from TTA to TCA, such that a PKHD1L1 point mutation rat is constructed and obtained. The rat model can be further applied in various research scenarios such as epilepsy pathogenesis, design of a novel anti-epileptic drug and the like. The present invention provides use of a method for detecting cortical excitability abnormity in detecting a phenotype of an epilepsy animal model. In the present invention, after the animal model is constructed and obtained, a somatosensory evoked potential method is further adopted to detect the presence of a phenotype of cortical excitability abnormity and thereby confirm whether the model is successfully constructed or not. The present invention can be used for detecting cortical excitability abnormity, and can also be used for verifying the effectiveness of a related drug or treatment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority of the Chinese patent application with the title of “Construction and Application of a Point Mutation Rat Epilepsy Model,” filed with the China National Intellectual Property Administration on December 6, 2022, Application No. 202211558984.6, and the Chinese patent application with the title of “A Method and Application for Identification of Cortical Excitability Abnormalities in an Animal Model of Epilepsy,” filed with the China National Intellectual Property Administration on June 28, 2023, Application No. 202310770803.4, both of which are incorporated herein by reference as if fully set forth herein. TECHNICAL FIELD

[0002] The invention belongs to the technical field of genetically modified animals, and in particular relates to the construction and application of a point mutation rat epilepsy model and a method and application of identifying abnormal cortical excitability in an epileptic animal model. BACKGROUND

[0003] Epilepsy is a serious chronic disease of the central nervous system, and is a temporary brain dysfunction caused by repeated attacks and highly synchronized abnormal discharge of brain neurons. It is clinically manifested by different disorders such as movement, sensation, consciousness, autonomic nerve and spirit, and is one of the more common diseases of the nervous system, affecting more than 65 million people. Disability, death and complications related to epilepsy have caused a heavy burden to patients and society. At present, little is known about the pathogenesis of epilepsy, and there is even significant heterogeneity in the pathogenesis considered by different scholars. Therefore, various animal models have been constructed to explain the pathogenesis of epilepsy. The animal models of epilepsy refer to specific species with an externally induced or inherited tendency to seizure. Therefore, in order to detect changes in electroencephalograms (EEGs), as well as behavioral characteristics in the state of epileptic seizures, a large number of epilepsy model studies have used inducible factors to keep epileptic seizures persistent. For example, one TLE animal model was constructed by intraperitoneally administering pilocarpine or pentylenetetrazole. Another animal model of TLE was established by injection of kainic acid into a lateral ventricle. However, the existing animal models of epilepsy cannot fully meet the clinical needs of epilepsy pathogenesis, pathophysiological process and treatment target screening. There are many kinds of animal models to explain the pathogenesis of epilepsy, so it is still unknown whether the constructed models can truly reflect the pathogenesis of epilepsy.

[0004] The detection of the constructed model is a necessary step, but there is no unified standard for detecting the successful construction of an animal epilepsy model in the existing data. For example, the common epilepsy model built with zebrafish needs to be detected by a zebrafish behavior meter, and the degree of epilepsy can be reflected according to the change of activity intensity of young zebrafish. In another example, in the Chinese patent CN201910244481.3 (entitled “A new method of drug-induced epilepsy model”), after drug induction, EEG detection of animal brainwaves, behavioral grading evaluation of epileptic rats, culture of primary hippocampal neurons in vitro, cell administration, and determination of the firing function of primary hippocampal neurons by a patch-clamp technique, it is necessary to determine whether the final rat model is epileptic.

[0005] It is known that an electrophysiological signal is the gold standard to evaluate the occurrence of epilepsy (or increased cortical excitability) and the effectiveness of drug intervention, but how to detect the corresponding animal model and / or its successful construction has become a technical problem to be solved. SUMMARY

[0006] One purpose of the invention is to provide a genetically modified (e.g., point mutant) rat, useful as a model for epilepsy, and methods for the construction and use / applications thereof. The mutant rat has a phenotype including increased neuronal excitability, which can simulate the phenotype of familial cortical myoclonic tremor with epilepsy (FCMTE) and other epilepsy patients. The mutant rat can also be useful for studying or determining the pathogenesis of epilepsy and for designing and testing novel anti-epileptic drugs.

[0007] Another purpose of the invention is to provide a method of and use for identifying cortical excitability abnormalities in an animal model of epilepsy. To determine whether the genetically modified rat was successfully constructed, a method using somatosensory evoked potential (SEP) to detect a phenotype including abnormal cortical excitability was created. This method can be extended to determine whether other animal models of epilepsy are successfully constructed.

[0008] The invention provides a method for constructing a genetically-modified rat (e.g., a rat model having a PKHD1L1 gene with a point mutation therein), including the following steps: designing single-guide RNA (sgRNA) using introns 22-23 and introns 24-25 of a PKHD1L1 gene as target sequences, annealing the sgRNA, ligating the annealed sgRNA into a plasmid vector with a T7 promoter, transcribing in vitro to obtain Cas9 / sgRNA, injecting the Cas9 / sgRNA and a targeting vector into fertilized rat eggs, and placing the fertilized rat eggs with the Cas9 / sgRNA and the targeting vector therein into a uterus of one or more pseudopregnant rat. The Fo generation rats are chimeric, with a mutation in the PKHD1L1 gene.

[0009] Preferably, the method includes amplifying introns 22-23 by a polymerase chain reaction (PCR) using primer pairs including PKHD1L1-5’ MSD-F and PKHD1L1-5’ MSD-R. The nucleotide sequence of PKHD1L1-5’ MSD-F is shown in SEQ ID NO: 1, and the nucleotide sequence of PKHD1L1-5’ MSD-R is shown as SEQ ID NO: 2. The method may further include amplifying introns 24-25 using primer pairs including PKHD1L1-3’ MSD-F and PKHD1LI-3’ MSD-R. The nucleotide sequence of PKHD1L1-3’ MSD-F is shown in SEQ ID NO: 3, and the nucleotide sequence of PKHD1L1-3’ MSD-R is shown in SEQ ID NO: 4.

[0010] Preferably, the PCR includes conditions of 94 °C for 5 min; 94 °C for 30 s, 62 °C for 30 s, 72 °C at 1 kb / min, a total of 30 cycles, and 72 °C for 10 min.

[0011] Preferably, the sgRNA has a sequence as shown in SEQ ID NO: 5 and / or SEQ ID NO: 6.

[0012] Preferably, the plasmid vector includes a pCS-3G vector.

[0013] Preferably, the targeting vector has a nucleotide sequence including or consisting of SEQ ID NO: 27.

[0014] Preferably, the method further comprises, after obtaining the Fo generation rats from at least one of the pseudopregnant rat, identifying the rat(s) having the PKHD1L1 gene point mutation chimeric by PCR.

[0015] When identifying the genetically modified rat using PKHD1L1-L-GT-F and PKHD1L1-L-GT-R, a 2662 bp sequence may be amplified. The nucleotide sequence of PKHD1L1-L-GT-F is shown as SEQ ID NO: 7, and the nucleotide sequence of PKHD1L1-L-GT-R is shown as SEQ ID NO: 8.

[0016] When identifying the genetically modified rat using PKHD1L1-R-GT-F and PKHD1L1-R-GT-R, a 2697 bp sequence may be amplified. The nucleotide sequence of PKHD1L1-R-GT-F is shown as SEQ ID NO: 9, and the nucleotide sequence of PKHD1L1-R-GT-R is shown as SEQ ID NO: 10.

[0017] Preferably, the PCR conditions when identifying the genetically modified rat include pre-denaturing at 94 °C for 2 min, denaturing at 98 °C for 10 s, annealing at 67 °C for 30 s, extending at 68 °C at 1 kb / min, repeating the denaturing, annealing, and extending for up to a total of 15 cycles, annealing at a temperature of -0.7 °C per cycle, denaturing again at 98 °C for 10 s, annealing again at 57 °C for 30 s, extending again at 68 °C at 1 kb / min, repeating the denaturing again, annealing again, and extending again for up to a total of 25 cycles, and finally extending the polynucleic acid containing the mutant PKHD1L1 gene at 68 °C for 10 min.

[0018] The invention also provides a method for constructing a stable genetically-modified rat, including mating the genetically-modified rat(s) with one or more wild-type rats, wherein the stable genetically-modified rat(s) are heterozygous. The heterozygote may be identified by genotype identification.

[0019] The invention also provides an application of a method of screening and / or developing epilepsy drugs using the genetically modified rat(s) or the stable genetically modified rat(s).

[0020] The invention provides an application of a method for determining the phenotype of a possibly epileptic animal or an epileptic animal model by detecting abnormal cortical excitability.

[0021] Preferably, detecting abnormal cortical excitability includes determining or detecting somatosensory evoked potentials.

[0022] The invention also provides a method of detecting a phenotype of an epileptic animal model or possibly epileptic animal, which comprises the following steps: fixing the head and limbs of the animal or model in a prone position, electrically stimulating a posterior tibial nerve by or through the skin, inserting or placing a recording needle and / or electrode into a subcutaneous region of the cranial roof, and inserting or placing a reference needle and / or electrode into the nose or a subcutaneous area above the nose.

[0023] The method of detecting the phenotypic of the epileptic animal may further comprise extracting a signal, filtering and amplifying the signal, measuring a peak latency according to a somatosensory evoked potential, evaluating a cortical excitability, and determining whether the animal is epileptic according to the cortical excitability.

[0024] Preferably, the posterior tibial nerve is electrically stimulated using a constant pressure square wave with a wave or pulse width of 0.1 ms and / or a frequency of 3 Hz. The constant pressure square wave has an intensity sufficient to cause the back toe to move or contract. A (further) subcutaneous needle in the back may be grounded.

[0025] Preferably, the method further comprises inputting the extracted signal (after filtering and amplification) into a computer or computer operating system for analysis (e.g., by determining an average superposition, for example with 1024 superposition times and an analysis duration of 56 ms). Finally, the method may further comprise displaying (and optionally printing) graphics including a pattern of the somosensory evoked potential. The peak latency may be measured using this somosensory evoked potential pattern.

[0026] Preferably, the epileptic animal includes a genetically modified epileptic rat or a genetically modified epileptic rat.

[0027] Beneficial effects of the invention include preliminary confirmation that the heterozygous mutation c.2602A>T in exon23 of the PKHD1L1 gene is a pathogenic mutation of the family by a pathogenicity study of a family with familial adult myoclonic epilepsy (see Fig. 17). The invention uses a CRISPR / Cas9 system to modify a PKHD1L1 gene to change a TTA sequence or codon to a TCA sequence or codon (e.g., knock in the P.L867S mutation in the rat PKHD1L1 gene), so as to construct a PKHD1L1 point mutation in a genetically modified animal. Three male PKHD1L1 point-mutated heterozygous (PKHD1L1+ / -) rats were observed for 5 consecutive days, and no spontaneous epilepsy was observed. However, PKHD1L1+ / - rats showed a phenotype of increased neuronal excitability, and a significantly lower threshold concentration of intraperitoneally injected pentylenetetrazole (PTZ) that induces epileptic seizure (e.g., in the PKHD1L1+ / - rats) than in wild-type rats, and the resting potential of brain electrophysiology of the PKHD1L1- / - rats was significantly lower than that of wild-type rats, enabling simulation of a FCMTE or other phenotype that may be characteristic of epileptic patients. The genetically-modified rat is useful for determining the pathogenesis of epilepsy, the design and testing of novel antiepileptic drugs, and various other research scenarios.

[0028] The invention provides a method for detecting abnormal cortical excitability to determine the phenotype of an epileptic or potentially epileptic animal. The method uses SEP to detect whether the constructed animal has an abnormal cortical excitability phenotype, so as to judge whether the construction is successful. The detection method of the invention can detect cortical excitability in rats, and the detected value can be used as an evaluation index for the efficacy of drugs that inhibit cortical excitability. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Fig. 1 shows the sequence of an exemplary PKDH1L1 gene mutation.

[0030] Fig. 2 is a diagram showing an exemplary construction strategy for a targeting vector useful in the present invention.

[0031] Fig. 3 shows the plasmid map of precut pCS-3G.

[0032] Fig. 4 shows the map of the targeting vector.

[0033] Fig. 5 is graph showing the activity detection results of various sgRNA sequences in accordance with the present invention.

[0034] Fig. 6 is a reproduction of a Southern blot showing the results of RNA electrophoresis, in which sgl refers to PKHDILl-sgRNAl, and sg2 refers to PKHDILl-sgRNAl 21

[0035] Fig. 7 is a reproduction of a Southern blot showing the results of Fo-generation identification by PCR using PKHD1L1-L-GT-F / PKHD1L1-L-GT-R in accordance with the present invention.

[0036] Fig. 8 is a reproduction of a Southern blot showing the results of Fo-generation identification by PCR using PKHD1L1-R-GT-F / PKHD1L1-R-GT-R in accordance with the present invention.

[0037] Fig. 9 is a reproduction of a Southern blot showing the results of Fi-generation identification by PCR using PKHD1L1-L-GT-F / PKHD1L1-L-GT-R in accordance with the present invention.

[0038] Fig. 10 is a reproduction of a Southern blot showing the results of Fi-generation identification by PCR using PKHD1L1-R-GT-F / PKHD1L1-R-GT-R in accordance with the present invention.

[0039] Fig. 11 is a reproduction of a Southern blot from an exemplary analysis of Fi generation positive rats in accordance with the present invention.

[0040] Fig. 12 shows an exemplary genotype sequencing analysis of wild-type, heterozygous and positive Fi generation rats in accordance with the present invention.

[0041] Fig. 13 shows the results of epileptic susceptibility determinations in wild-type rats and PKHDILI’ rats in accordance with the present invention.

[0042] Fig. 14 shows the effect of H89 on sEPSC / sIPSC measurements of cortical pyramidal neurons, in which A shows sEPSC currents of pyramidal neurons in groups of control, epileptic and treated rats, B shows the sEPSC amplitudes in pyramidal neurons in the rats, C shows the sEPSC frequencies in pyramidal neurons in the rats, D shows sIPSC currents in pyramidal neurons of each group of rats; E shows sIPSC amplitudes in the pyramidal neurons in the rats; and F shows sIPSC frequencies in the pyramidal neurons in the rats (3 rats in each group and 6 cells in each group, * refers to P <0.05, and ** refers to P <0.01).

[0043] Fig. 15 shows the effect of H89 on the Na+ and K+ currents in the cerebral cortex of the control, epileptic and treated groups of rats, wherein A shows the Na+ currents in the pyramidal neurons of the rats, and B shows the K+ currents in the pyramidal neurons of the rats (3 rats in each group, 6 cells in each group, P <0.05).

[0044] Fig. 16 shows the effect of H89 on the action potential of pyramidal neurons in the cerebral cortex of the control, epileptic and treated rats, wherein A are waveforms of the action potential of pyramidal neurons in the cerebral cortex of rats in each group, B are sample single action potentials of pyramidal neurons in each group of rats, C is a graph showing the action potential threshold of the pyramidal neurons in each group of rats, D is a graph showing the positive post-potential recovery of the action potential in each group of rats, E is a graph showing the half-duration of the action potential in each group of rats, and F is a graph showing the peak action potential in each group of rats (3 rats per group, 6 cells in each group, and * refers to P <0.05).

[0045] FIG. 17 is a diagram of an exemplary family with a history of familial adult myoclonic epilepsy. DETAILED DESCRIPTION OF EMBODIMENTS

[0046] The invention concerns a method for constructing a genetically-modified rat having a PKHD1L1 gene with a mutation therein, including the following steps: designing single-guide RNA (sgRNA) using introns 22-23 and introns 24-25 of a PKHD1L1 gene as target sequences, annealing the sgRNA, ligating the annealed sgRNA to a plasmid vector with a T7 promoter, and transcribing the plasmid vector in vitro to obtain Cas9 / sgRNA. The Cas9 / sgRNA and a targeting vector are injected into fertilized rat eggs to obtain gene-edited fertilized eggs, and the gene-edited fertilized eggs are placed or inserted into a uterus of one or more pseudopregnant rat to obtain Fo generation, chimeric rats with the mutant PKHD1L1 therein.

[0047] The PKHD1L1 gene in the invention is preferably selected from a family with a history of familial adult myoclonic epilepsy. A family diagram showing such a family is shown in Fig. 17. The family has a total of 30 people in five generations and 6 patients with familial adult myoclonic epilepsy (identified in black), which conforms to the characteristics of autosomal dominant inheritance. All patients had myoclonic seizures with or without generalized tonic-clonic seizures and subtle tremors at the distal extremity. The onset of seizures was adult. Electroencephalographic examination (EEG) indicated bilateral symmetric spinoid-slow wave release, and evoked potential testing showed positive giant potential(s) and C-reflex. Anti-epileptic drug treatment effectively controlled the seizures, and the disease course was benign. All members of the family were effectively diagnosed and their clinical phenotypes were consistent. By whole genome exon sequencing combined with linkage analysis, it was found that 5 patients (1 of the 6 patients had died, as indicated by the slash through the black square in Fig. 17) had heterozygous mutations of the PKHDI LI gene exon 23:c.2602A>T, and 11 family controls were homozygous with co-segregation. The PKHD1L1 gene was further screened in 246 cases (e.g., persons) from a normal population, matched by age, sex, region and ethnicity, and did not find the mutation in the normal population. Therefore, the heterozygous mutation of the PKHDI LI gene exon 23:c.2602A>T (see Fig. 1) was preliminarily confirmed as a pathogenic mutation in the family.

[0048] The PKHD1L1 gene that encodes the polycystic kidney and hepatic disease 1-like protein 1 is on the positive chain of chromosome 7, and has a total length of about 172.46 kb. The Gene ID of this gene in Rattus norvegicus (Norway rats) is 314917. In one example, a PKHD1L1-201 transcript (ENSRNOT00000005958.7, NM 001034931; referred to hereinafter as “the example PKHD1L1 gene”) was used to conduct a point mutation study in rats. Specifically, amino acid Lys at 867 of the example PKHD1L1 gene was mutated to Ser by a CRISPR / Cas9 system, the corresponding base was changed from TTA to TCA, and the sgRNA was designed using intron(s) 22-23 and intron(s) 24-25 (Fig. 2).

[0049] In one embodiment, in order to ensure the efficiency of the designed Cas9 / sgRNA, PCR amplification and sequencing verification was performed on the singlestranded DNA (SD) rat target site sequence to ensure that the sgRNA recognition sequence is completely consistent with the SD rat DNA sequence. When the primers included PKHD1L1-5’ MSD-F (SEQ ID NO: 1) and PKHD1L1-5’ MSD-R (SEQ ID NO: 2), the amplified product has a length of 754 bp. When the primers included PKHD1L1-3’ MSD-F (SEQ ID NO: 3) and PKHD1L1-3’ MSD-R (SEQ ID NO: 4), the amplification product has a length of 569 bp. The PCR amplification procedure and conditions preferably include a temperature of 94 °C for 5 min; a temperature of 94 °C for another 30 s, a temperature of 62 °C for 30 s, a temperature of 72 °C at 1 kb / min, carried out for 30 cycles; and a temperature of 72 °C for 10 min. The PCR products were sequenced, and the results showed that the SD rat target sequence was completely consistent with the sequence obtained from Genebank and Ensembl, and are suitable for point mutated sgRNA target genes. Table 1: Primers for amplification of rat target sequences by PCR Primer Sequence (5’-3’) SEQ ID NO Tin (°C) Prod, size (bp) PKHD1L1-5’ MSD-F GCATCAAGCTTGGTACCGATAATCACAAGACACAATAGACGCAGA 1 61 754 PKHD1L1-5’ MSD-R ACTTAATCGTGGAGGATGATCTTGCTTCACAAACAAAGGGACCTG 2 63 PKHD1L1-3’ MSD-F GCATCAAGCTTGGTACCGATGACAGAAGCAAAGCCAGAGAATAAA 3 60 569 PKHD1L1-3’ MSD-R ACTTAATCGTGGAGGATGATGAAGTTTACATAACTCAAGCAGTCCA 4 60

[0050] Two sgRNAs were designed and made based on the target gene(s), and the sgRNAs are specifically PKHDIL 1-sgRNA 1 (SEQ ID NO: 5) and PKHDlLl-sgRNA12 (SEQ ID NO: 6; see Table 2). The sgRNA(s) are optimally connected to a pCS-3G carrier (shown in Fig. 3) by annealing polymerization, and the connected product is converted into a sample for sequencing verification (e.g., Cas9 / sgRNA that can be microinjected). The annealing polymerization preferably includes annealing at 65 °C for 5 min. Table 2: sgRNA sequence sgRNA GuideRNA sequence SEQ ID NO PKHD 1L1-sgRNA 1 GGTAGGCTAGACTTTAA 5 PKHDlLl-sgRNA2 GGCCTTCGTATTAGCTATA 11 PKHDlLl-sgRNA3 GGAATCCCTATAGCTAATACGA 12 PKHDlLl-sgRNA4 GGCTACTATGTTAAATATG 13 PKHDlLl-sgRNA5 GGAGTCTTAAAGTGAACC 14 PKHDlLl-sgRNA6 GGGAAGTTAAGAAACACAAT 15 PKHDlLl-sgRNA7 GGCACCTGTGGGCATGTGTAGA 16 PKHDlLl-sgRNA8 GGTCACCCTTAAGCCCCCAAAA 17 PKHDlLl-sgRNA9 GGTCTACATGTATGTCACCACC 18 PKHDILl-sgRNAlO GGTTAATTTAGTCATGTATA 19 PKHD1L1 -sgRNA 11 GGTCAAATAACTAGAACTGC 20 PKHDlLl-sgRNA12 GGA A AGTTGAGTGTTrC A xJ vJ / iZiZi xJ JL JL VJ / WJ JL xJ JL JL v / V / Ti 6 PKHD IL 1-sgRNA 13 GGAAGTTATTTGTTATGAA 21 PKHD IL 1-sgRNA 14 GGAAATCTGGGTCCTTAGAA 22

[0051] The plasmid profile of the targeting vector for microinjection is shown in Fig. 4, and the nucleotide sequence of the targeting vector is shown in SEQ ID NO: 27.

[0052] Cas9 / sgRNA and the targeting vector were microinjected into fertilized eggs of rats, and the Fo rats were bom after the injection. The Fo rats obtained were chimeras due to the rapid cleavage rate of the embryos in the early stage, and the chimeric rats were also identified by PCR after the Fo generation. Chimeric rats are positive for both PKHDIL1-L-GT-F / PKHD1L1-L-GT-R (2662 bp) and PKHD1L1-R-GT-F / PKHD1L1-R-GT-R (2697 bp). Preferably, PCR identification uses a Touchdown mode, and constructs the reaction system according to the instruction manual for KOD-FX enzyme. The PCR identification procedure preferably includes: pre-denaturing at 94 °C for 2 min; denaturing at 98 °C for 10 s, annealing at 67 °C for 30 s, extending at 68 °C at 1 kb / min, and repeating the denaturing, annealing, and extending steps for a total of 15 cycles, annealing at a temperature of -0.7 °C per cycle; a second denaturing at 98 °C for 10 s, a second annealing at 57 °C for 30 s, a second extending at 68 °C at 1 kb / min, and repeating the second denaturing, annealing, and extending steps for a total of 25 cycles; and finally extending at 68 °C for 10 min. Table 3: Primers for identification of point mutation chimera Primer Sequence (5’-3’) SEQ ID NO Tm (°C) Product size (bp) PKHD1L1-L-GT-F tgcagatgttgtgagaaaagcaagaca 7 60 Mut: 2662 PKHD1L1-L-GT-R ccagtcttgatgttttatagatacttcccc 8 59 WT: 2650 PKHD1L1-R-GT-F tccaatccatttatgtggatgccgtgt 9 62 Mut: 2697 PKHD1L1-R-GT-R aggatgcttgaatctttcttctaagggg 10 60 WT: 2680

[0053] The invention also concerns a method for constructing a stable genetically modified rat with a point mutation in a PKHD1L1 gene, including the following steps: mating an Fo generation chimeric rat with the point mutation with a wild-type rat to produce an Fi generation including the stable genetically modified rat with the point mutation in the PKHD1L1 gene. The Fi generation rat with the PKHDILI gene pOint mutation is genetically stable and heterozygous.

[0054] In the present invention, the Fo generation genotype-positive rat is selected to mate with one or more wild-type rats to obtain the Fi generation rat with a stable genotype. The genotype of the Fi generation rats is identified by PCR, Southern blot analysis and sequencing, in which the PCR for identification is the same or substantially the same as that for constructing and / or determining the Fo generation, and it will not be repeated here. EcoRV and Spel were used as restriction enzyme sites for Southern blot analysis. Correct recombination was determined and / or detected using 3’ Probe-A. When recombination is correct, two bands, wild-type and mutant type, appear. Random insertion was determined and / or detected using LR Probe-A. When no random insertion is detected, two bands, wildtype and mutant type, appear. Table 4: Primer information of 3’ Probe-A and LR Probe-A Primer Sequence (5’-3’) Product size (bp) Tm (°C) SEQID NO PKHD1L1 -LR-Probe-A-F aggatctctggccaacttcattgg 470 60 23 PKHD1L1 -LR-Probe-A-R ttctgtttctaatgttagtggaaatgc 55 24 PKHDlLl-3’Probe-A-F ccagtccccagaacaattggctaga 546 61 25 PKHD 1L1 -3 ’Probe-A-R actgtgtggaggcaaagaagcatga 61 26

[0055] For Fi and successive generations with point mutations, heterozygous and homozygous genotypes can also be detected by PCR validation and sequencing. The primers used for this detection include PKHD1L1-R-GT-F and PKHD1L1-L-GT-R, and the optimized conditions include a first stage at a temperature of 94 °C for 5 min, a cycling stage including a temperature of 94 °C for 30 s, a temperature of 62 °C for 30 s, a temperature of 72 °C at 1 kb / min, and a total of 30 cycles; and a final stage at a temperature of 72 °C for 10 min. Since the mutated and the wild-type sequences are both 625 bp, the homozygous, heterozygous and wild-type genotypes are confirmed by sequencing.

[0056] The invention also concerns methods of screening and / or developing epilepsy drugs, using a genetically modified rat having a PKHD1L1 gene with a point mutation therein or a stable genetically modified rat with the point mutation in the PKHD1L1 gene.

[0057] The invention also provides a method of or application for detecting abnormal cortical excitability and / or detecting or determining a phenotype of an epileptic or possibly epileptic animal.

[0058] The method of detecting abnormal cortical excitability preferably includes determining (e.g., testing or obtaining) a somatosensory evoked potential (SEP) in the animal. There is no special limitation on the specific construction method or animal, although the animal is preferably a rat, mouse or zebrafish. A genetically-modified rat is an example and / or embodiment of the invention, but the invention is not limited to genetically-modified rats. A mutation in the PKHD1L1 gene exon 23:c.2602A>T (which may be heterozygous) was confirmed as a pathogenic mutation in the family shown in Fig. 17 through a pathogenicity study of familial adult myoclonic epilepsy (FAME) in the family, and in one example, a CRISPR / Cas9 system was used to knock in the P L867S mutation into the PKHD1L1 gene to change the corresponding codon from TTA to TCA, resulting in the construction of a PKHD1L1 point-mutant rat. An electrophysiological examination of the epileptic seizure patients in the family found increased cortical excitability, and the seizures were effectively controlled by anti-epileptic drug treatment. At the same time, the genetically modified rats having or expressing the mutant PKHD1L1 gene constructed in accordance with the invention showed significantly shorter incubation periods and increased SEP amplitudes, compared with wild-type rats with matching body weight, suggesting that the cortical excitability of PKHD1L1+ / - rats was significantly higher than that of wild-type rats. This study further verified the increased cortical excitability of PKHD1L1+ / - rats, which can better simulate the FAME phenotype and phenotypes of other epilepsy patients, and can be further applied to research into the pathogenesis of epilepsy and the design and testing of new anti-epileptic drugs.

[0059] The invention concerns a method for detecting a phenotype in an epileptic or possibly epileptic animal (e.g., an epileptic animal model), which comprises the following steps: fixing the head and limbs of the animal in a prone position, electrically stimulating the posterior tibial nerve of the ankle of a hind limb (e.g., the right hind limb) through the skin, subcutaneously placing a recording needle and / or electrode into the Cz region of the cranial roof, and subcutaneously placing a reference needle and / or electrode into an area above the nose.

[0060] The method for detecting a phenotype in an epileptic or possibly epileptic animal may further comprise extracting a signal. After the extracted signal is filtered and amplified, an input system measures the peak latency according to one or more somatosensory evoked potentials, evaluates a cortical excitability, and determines whether the animal is epileptic according to the cortical excitability.

[0061] Electrically stimulating the posterior tibial nerve may include transmitting a constant pressure square wave with a pulse or wave width of 0.1 ms and a frequency of 3 Hz. The intensity of the square wave may be sufficient to cause movement (e.g., micro-movement) in the back toe. Electrically stimulating the posterior tibial nerve may further include subcutaneously placing a needle in the back of the animal, and the needle may be grounded. The method for detecting a phenotype in an animal optimally filters and amplifies the extracted signal, and inputs it into a computer operating system for determining an average superposition. The average superposition may be determined from 1024 instances, the analysis time may be 56 ms, and the computer operation system may be configured to display and print a pattern of somosensory evoked potentials to determine or measure a peak latency.

[0062] In order to further illustrate the invention, the construction of a genetically-modified, mutant rat as a model for epilepsy and an application thereof are described in detail in combination with the accompanying drawings and below examples, but the invention is not limited to the drawings or examples. Example 1: Screening for disease-causing mutated genes

[0063] In the family shown in Fig. 17 (Family 1), there are 30 people in five generations, of whom 6 had the disease (e.g., epilepsy), all 6 of which were consistent with autosomal dominant inheritance. All 6 patients had myoclonic seizures with or without generalized tonic-clonic seizures, and with or without subtle tremors at the distal extremity. The onset of seizures was adult, and electrophysiological examination showed increased cortical excitability. The seizures were effectively controlled by anti-epileptic drug treatment, and the course of the disease was benign. All patients in Family 1 were diagnosed clearly, and their clinical phenotypes were consistent. Whole genome exon sequencing combined with linkage analysis showed that 5 patients (1 of the 6 patients had died) had heterozygous mutations in PKHD1L1 gene exon 23:c.2602A>T, and 11 controls were homozygous with co-segregation.

[0064] The PKHD1L1 gene was screened in 246 normal people matched by age, sex, region, and ethnicity, and the mutation site could not be found in the normal population, so it was preliminarily confirmed by genetics that this site in PKHD1L1 was a pathogenic mutation in this family. Table 1: Clinical data of Family 1 patients ri Age (Yrs) Onset2 Seizure t3 P4 SEP5 C6 AEDs Cure7 type HIM D8 58 GTC + NA NA NA PHT N° seizure 1 G: Gender 2 Age of patient in years at time of disease onset. 3 T: tremor 4 P: Polycystic kidney / poly cystic liver 5 SEP: Upper limb SEP (P25-N 30), in pV. 6C: C reflex 7 Cure: AED Curative effect. 8 D: Deceased G1 Age (Yrs) Onset2 Seizure type T3 P4 SEP5 C6 AEDs Cure7 III3 F 59 40 M9+ GTC + NA 38.0 + PHT No seizure III7 F 52 38 M+ GTC + NA 75.5 + PB and PHT No seizure 1119 M 49 30 M+ GTC + NA 19.04 + PB and PHT No seizure IV6 F 36 35 M+ GTC — NA 22.28 + PHT No seizure IV1 1 M 24 11 M+ OTC + NA 11.97 + VPA No seizure

[0065] Preparation of the genetically-modified rat in knock-in mode using a PKHD1L1 gene (Gene ID: 314917, Pkhdlll-201 transcript ENSRNOT00000005958.7, NM00I034931):

[0066] 1. Cas9 / sgRNA design and construction

[0067] 1.1 Cas9 / sgRNA design

[0068] Based on sgRNA design principles, 7 sgRNAs were designed in the 5’ target site and 3’ target site area, respectively (Table 1).

[0069] 1.2 Construction of Cas9 / sgRNA plasmid

[0070] The sgRNA sequence synthesis primers shown in Table 1 were designed and connected to the pCS-3G carrier shown in FIG. 3 by annealing polymerization (65 °C, 5 min). After the conversion of the connected product, the sample was sent for sequencing verification. 9 M: Myoclonus

[0071] The CRISPR / Cas9 activity detection method developed by Biocytogen-UCATM was used to detect sgRNA activity, and the results are shown in A-B of Fig. 5. PKHDILl-sgRNAl (Guide #1) and PKHD1LI -sgRNA 12 (Guide #12) were selected to carry out the next experiment.

[0072] 1.3 RNA preparation of sgRNA

[0073] PKHDILl-sgRNAl and PKHDlLl-sgRNA12 were connected to plasmid vectors with a T7 promoter and transcribed in vitro to obtain RNA for microinjection (Fig. 6).

[0074] 1.4 Construct the targeting vector shown in Fig. 4.

[0075] 1.5 Microinjection of Cas9 / sgRNA

[0076] Cas9 / sgRNA and the targeting vector were microinjected into fertilized rat eggs. Data for the Fo rats at / after birth is shown in Table 5. Table 5: Birth statistics of Fo rats Date Family Number of transferred zygotes Due date Number of births Positive number 2018 / 01 / 12 SD 210 2018 / 02 / 03 41 0 2018 / 03 / 30 SD 250 2018 / 04 / 21 35 2 2018 / 04 / 08 SD 328 2018 / 04 / 30 44 5

[0077] 1.6 Genotype detection of Fo generation rats

[0078] Primers PKHD1L1-L-GT-F / PKHD1L1-L-GT-R (Mut: 2662 bp, WT: 2650 bp) and PKHD1L1 -R-GT-F / PKHD1L1 -R-GT-R (Mut: 2697 bp, WT: 2680 bp) were identified by PCR, and the genotype of the Fo generation rats was conventionally determined. The results are shown in Figs. 7 and 8. The PCR products and sequencing showed that EY55-072 and EY55-073 are positive Fo rats.

[0079] 1.7 Genotypes and Southern blot identification of Fi generation rats

[0080] An Fo generation rat having a positive genotype were selected to mate with a wild-type rat to obtain Fi generation rats with a stable positive genotype. The mating results are shown in Table 6. Table 6: Mating statistics Rat OD Mating date Maturity date Birth number No. Positive EY55-073 (¢) 2018 / 05 / 24 2018 / 06 / 14 24 10

[0081] 1.7.1 Genotype identification of Fi generation (the primer design was as shown for the F0 generation):

[0082] The primers for identification of Fi generation rats are the same and / or were designed according to the same procedure(s) as those in the method of genotype identification and / or detection in the Fo generation. The results are shown in part in Figs. 9 and 10. PCR identification and point mutation site sequencing are used to obtain the results. The results indicated that 1EY55-025, 1EY55-027, 1EY55-029, 1EY55-030, 1EY55-031, 1EY55-032, 1EY55-034, 1EY55-035, 1EY55-036, 1EY55-037 and 1EY55-038 were positive Fi generation rats.

[0083] 1.7.2 Southern blot analysis of Fi generation positive rats

[0084] The DNA of Fi generation rats (e.g., obtained from the tail) identified as positive by PCR was extracted and tested by Southern blotting and sequencing. The test results are shown in Fig. 11, and show that 1EY55-025, 1EY55-027, 1EY55-029, 1EY55-030, 1EY55-031, 1EY55-032, 1EY55-034, 1EY55-036, 1EY55-037 and 1EY55-038 are correctly reassembled and do not have a random insertion.

[0085] 1.7.3 Genotyping analysis of positive Fi rats with correct recombination and no random insertion

[0086] Primers PKHD1L1-R-GT-F and PKHD1L1-L-GT-R were used for PCR validation and sequencing on positive Fi generation rats with correct recombination and no random insertions. The results are shown in Fig. 12, where “Mut / Mut” refers to a homozygous genotype, “Mut / +” refers to a heterozygous genotype, and “+ / +” refers to a wild-type genotype. Example 2

[0087] The epileptic behavior and phenotype(s) of rats having the PKHD1L1 gene with the point mutation constructed in Example 1 were analyzed.

[0088] Three male heterozygous rats having the PKHD1L1 gene with the point mutation (PKHD1L1+ / -) were first observed for 5 consecutive days for spontaneous epilepsy. However, no spontaneous epileptic behavior was observed.

[0089] Subsequently, the epileptic susceptibility of PKHD1L1+ / - rats was studied using a PTZ-induced epilepsy model: A total of 10 male PKHD1L1+ / - rats and 10 male wildtype (WT) rats with body weights matched with age (in weeks) were selected for inducing epileptic seizures with PTZ (40 mg / kg). The dose of PTZ selected is lower than the conventional dose in such animal modeling. Only 2 out of the 10 wild-type rats (20%), but 7 out of the 10 (70%) PKHD1L1+ / - rats, were induced to have grand mal seizures of grade 4-5 (see A in Fig. 13). The maximum seizure level reached by the PKHD1L1- / - rats was significantly higher than that of the wild-type rats (see B in Fig. 13). Further, additional PTZ injections (5 mg / kg at 15-minute intervals) were administered in rats that did not reach grade 4-5. The mean dose of PTZ required for wild-type rats to exhibit such seizures was statistically significantly higher than that required for PKHD1L1+ / - rats (see C in Fig. 13). These results indicate that PKHD1L1+ / - rats having a PKHDIL1 gene with the c.2602A>T point mutation have seizure susceptibility characteristics.

[0090] The PKHD1L1+ / - rats were further tested by SEP to detect a phenotype corresponding to abnormal cortical excitability. The specific methods are as follows.

[0091] The head and limbs of PKHD1L1+ / - rats were fixed in a prone position, and the posterior tibialis nerve of the ankle of the right hind limb was stimulated by electrocutaneous stimulation. The stimulation parameters included a constant pressure square wave having a wave or pulse width of 0.1 ms and a frequency of 3 Hz. The intensity of the stimulation was regulated and / or adjusted to cause posterior toe micromovement. A subcutaneous needle on the back was grounded. The recording needle / electrode was inserted subcutaneously into the Cz region at the top of the skull, and the reference needle / electrode was inserted subcutaneously above the nose. The signal extracted from the recording needle / electrode was filtered and amplified, and input into a computer for determining an average superposition of 1024 superposition times, using an analysis time of 56 ms. The resulting somosensory evoked potential pattern was displayed and printed to measure its peak latency. Waves are labelled according to their polarity and order of occurrence (Pl, P2,......Nl, N2, where P is a positive wave and N is a negative wave). The number indicates the number of the wave in the order in which the waves appear.

[0092] The results are shown in Table 7. Compared with wild-type (WT) rats with matched body weight, PKHD1L1+ / - (MU) rats had a significantly shorter incubation period of SEP (10.17 ± 1.17, vs. 12.32 ± 1.65, P = 0.0071), and the amplitude of SEP tended to increase (3.95 ± 1.72, vs. 2.87 ± 1.6, P = 0.1794), indicating that the cortical excitability of PKHD1L1+ / - rats was significantly higher than that of wild-type rats. This study further verified the increased cortical excitability of PKHD1L1+ / - rats, which better simulate the phenotype of FAME patients and other epilepsy patients. Table 7: Latency and amplitude of SEP in WT and MU rats No. Gender Weight (g) Group P40-N50 Wave amplitude (pV) P40 Latency (ms) 20230304001 M 244 WT 1.76 13.6 20230304002 F 226 WT 2.4 10.5 20230304003 F 273 WT 4 10.4 20230304004 F 261 WT 5.4 10.9 20230304005 M 428 WT 3.3 13.8 20230317006 F 308 WT 4.2 14 20230317007 M 475 WT 2.2 13.9 20230317008 M 477 WT 2.1 13.5 20230317009 M 480 WT 3.2 14.8 20230317010 M 487 MU 6.8 10 20230330011 M 385 WT 5.6 11.5 20230330012 F 365 WT 0.3 11.7 20230330013 M 586 MU 1.92 10.9 20230330014 M 572 MU 4 9.7 20230330015 M 608 MU 2.1 12.5 20230403016 F 342 MU 3.4 9.5 20230403017 F 320 MU 5.3 9.1 20230403018 F 315 MU 4.1 9.5 20230428019 F 403.9 WT 1.8 10.1 20230428020 M 495.8 WT 1.05 11.4 Example 3

[0093] Results of electrophysiological examination of transgenic animal brain slices

[0094] 1. In transgenic animals (PKHD1L1+ / - rats), neuronal excitability increased, and H89 reduced the frequency of spontaneous excitatory post synaptic currents (sEPSC) in pyramidal neurons in the cerebral cortex.

[0095] A patch clamp technique was used to record sEPSC and sIPSC (e.g., spontaneous inhibitory post synaptic currents) of cortical pyramidal neurons in the computer, and to observe the changes in excitatory synaptic transmission and inhibitory synaptic transmission of pyramidal neurons in each group of rats, so as to reflect any changes in neuronal excitability. It was found that the sEPSC amplitude and frequency were higher in the epilepsy group (e.g., the PKHD1L1+ / - rats) than in the control group (e.g., the wild-type rats), and the amplitude and frequency (e.g., in sEPSC) decreased after H89 treatment. Experimental results also showed that excitability was increased in the epileptic group (e.g., the PKHD1L1+ / - rats), and H89 could be used to reduce the amplitude and frequency of cortical sEPSC and reduce its excitability (e.g., in the PKHD1L1+ / - rats), thus playing a therapeutic role (see Fig. 14).

[0096] 2 H89 does not affect the excitability of pyramidal neurons (and optionally affect Na+ and K+ currents) in the cerebral cortex. Neuronal excitability is related to Na+ and K currents, so the Na+ and K+ currents in pyramidal neurons in the cerebral cortex of each group of rats (e.g., PKHD1L1+ / - and wild-type) were recorded. As shown in Fig. 15, there was no difference in Na+ and K+ currents in pyramidal neurons of rats among all groups (3 rat in each group, 6 cells in the control [WT] group, 6 cells in the epilepsy [e.g., PKHD1L1+ / -] group, and 6 cells in the epilepsy + H89 group), P <0.05.

[0097] 3 H89 can reduce the excitability of pyramidal neurons in the cerebral cortex of rats with epilepsy.

[0098] In order to further confirm the excitability of pyramidal neurons in the cerebral cortex, the action potential of pyramidal neurons was recorded. Action potential can intuitively reflect the excitability of neurons. The recorded action potentials of pyramidal neurons showed that the action potential threshold of rats in the epilepsy group (e.g., PKHD1L1- / - rats) decreased (P <0.05), but the action potential threshold increased after H89 treatment (P <0.05). Compared with the control group (e.g., WT rats), the positive potential of the epileptic group was decreased after H89 treatment, and the neuronal potential was increased after H89 treatment (P >0.05). There was no difference in the peak and half-duration of the action potential between the two groups. A decrease in positive post-potential of pyramidal neurons in epileptic rats indicated that the function of the sodium ion pump was weakened. It was shown that a reduced neuron action potential threshold in epileptic rats indicates decreased excitability of pyramidal neurons, and H89 reversed this trend and improved epileptic symptoms in rats (Fig. 16).

[0100] Although the above embodiments give a detailed description of the invention, they are only part of the embodiments of the invention, not all embodiments, and other embodiments can be obtained according to the embodiments without creativity, which are within the scope of protection of the invention.

Claims

1. A method for constructing a rat model with a PKHD1L1 gene point mutation, comprising designing single-guide RNA (sgRNA) using introns 22-23 and introns 24-25 of a PKHD1L1 gene as target sequences, annealing the sgRNA, ligating the sgRNA into a plasmid vector with a T7 promoter, transcribing the plasmid vector in vitro to obtain Cas9 / sgRNA, injecting the Cas9 / sgRNA and a targeting vector into fertilized rat eggs to obtain gene-edited fertilized eggs, and placing the gene-edited fertilized eggs in a uterus of pseudopregnant rat to obtain Fo generation rats having a PKHD1L1 gene point mutation chimeric.

2. The method described in claim 1, further comprising amplifying introns 22-23 by a PCR using primer pairs including PKHD1L1-5’ MSD-F and PKHD1L1-5’ MSD-R, wherein a nucleotide sequence of PKHD1L1-5’ MSD-F is shown in SEQ ID NO: 1 and a nucleotide sequence of PKHD1L1-5’ MSD-R is shown in SEQ ID NO: 2, and amplifying introns 24-25 by the PCR using primer pairs including PKHD1L1-3’ MSD-F and PKHD1L1-3’ MSD-R, wherein a nucleotide sequence of PKHD1L1-3’ MSD-F is shown in SEQ ID NO: 3 and a nucleotide sequence of PKHD1L1-3’ MSD-R is shown in SEQ ID NO: 4.

3. The method described in claim 2, wherein the PCR includes conditions of 94 °C for 5 min; 94 °C for 30 s, 62 °C for 30 s, 72 °C at 1 kb / min, 30 cycles, and 72 °C for 10 min.

4. The method described in claim 1, wherein the sgRNA has a sequence as shown in SEQ ID NO: 5 and SEQ ID NO: 6.

5. The method described in claim 1, wherein the plasmid vector with the T7 promoter includes a pCS-3G vector.

6. The method described in claim 1 or 4 or 5, wherein the sgRNA is ligated into the pCS-3G vector by annealing polymerization to form a connected product, and the connected product is transcribed in vitro to obtain the Cas9 / sgRNA.

7. The method described in claim 1, wherein the targeting vector has a nucleotide sequence shown as SEQ ID NO: 27.

8. The method described in claim 1, further comprising, after obtaining the Fo generation rats, identifying the rats having the PKHD1L1 gene point mutation chimeric by PCR, wherein the Fo generation rats having the PKHD1L1 gene point mutation chimeric include: when using PKHDI LI-L-GT-F (SEQ ID NO: 7) and PKHD1L1-L-GT-R (SEQ ID NO: 8) for PCR identification, amplifying a 2662 bp sequence, and when using PKHD1L1-R-GT-F (SEQ ID NO: 9) and PKHD1L1-R-GT-R (SEQ ID NO: 10) for PCR identification, amplifying a 2697 bp sequence.

9. The method described in claim 8, wherein the PCR for identifying the rats having the PKHDIL1 gene point mutation chimeric includes pre-denaturing at 94 °C for 2 min; denaturing at 98 °C for 10 s, annealing at 67 °C for 30 s, extending at 68 °C at 1 kb / min, 15 cycles, annealing at a temperature of -0.7 °C per cycle; denaturing at 98 °C for 10 s, annealing at 57 °C for 30 s, extending at 68 °C at 1 kb / min, 25 cycles; and extending at 68 °C for 10 min.

10. A PKHDI LI gene point mutation rat model, obtained by the method described in one of claims 1-9.

11. A method for constructing a stable genetic PKHD1L1 gene point mutation rat model, comprising mating the Fo generation rat having the PKHD1L1 gene point mutation chimeric obtained by the method described in one of claims 1 to 9 with wild-type rats, wherein the stable genetic PKHD1L1 gene point mutation rat model is a heterozygote of an Fi generation.

12. The method described in claim 11, further comprising identifying the heterozygote of the Fi generation by genotype identification.

13. The method described in claim 12, wherein the genotype identification includes PCR identification, Southern blot identification, or sequencing identification.

14. A stable genetic PKHD1L1 gene point mutation rat model, obtained by the method described in one of claims 11 to 13.

15. An application of the PKHDILI gene point mutation rat model described in claim 10 or the stable genetic PKHDILI gene point mutation rat model described in claim 14 in screening and / or developing one or more epilepsy drugs.

16. A method of detecting cortical excitability abnormalities in a phenotype of the PKHD1L1 gene point mutation rat model described in claim 10 or the stable genetic PKHD1L1 gene point mutation rat model described in claim 14.

17. The method of described in claim 16, wherein the method for detecting cortical excitability abnormalities includes somatosensory evoked potentials.

18. A method of detecting a phenotype in an epileptic animal model, comprising fixing a head and limbs of the epileptic animal model in a prone position, electrically stimulating a posterior tibial nerve by the skin, inserting a recording needle electrode into a subcutaneous region of a Cz region of a cranial roof, inserting a reference needle electrode into a subcutaneous region above a nose, filtering and amplifying an extracted signal, inputting the extracted signal to a system, measuring a peak latency according to an output somatosensory evoked potential, evaluating a cortical excitability, and determining whether the animal model is epileptic according to the cortical excitability.

19. The method described in claim 18, wherein the electrical stimulation includes parameters comprising a constant pressure square wave having a wave width of 0.1 ms, a frequency of 3 Hz, and an intensity sufficient to cause back toe micromovement, and a subcutaneous needle is grounded on a back.

20. The method described in claim 18, wherein after the extracted signal is filtered and amplified, the extracted signal is input to the system to measure the peak latency according to the output somatosensory evoked potential by inputting the filtered and amplified extracted signal to a computer operating system for average superposition, wherein a number of superposition times is 1024, an analysis time is 56 ms, and the system displays and prints somatosensory evoked potential graphics to measure the peak latency.

21. The method described in Claim 18, wherein the epileptic animal model includes a rat epileptic model or a mouse epileptic model.

22. The method described in Claim 21, wherein the epilepsy animal model is the PKHD1L1 gene point mutation rat model described in claim 10 or the PKHD1L1 gene point mutation rat model described in claim 14.INTERNATIONAL SEARCH REPORT International application No. PCT / CN2023 / 120325A. CLASSIFICATION OF SUBJECT MATTER C12N 15 / 85(2006.01)i; C12N 15 / 113(2010.01)i; C12N 15 / 89(2006.01)i; A01K 67 / 027(2006.01)i; A61K 49 / 00(2006.01)i; A01K 15 / 02(2006.01)i; A01K 67 / 02(2006.01)i According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) IPC: C12NA01KA61K Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) CNABS: CNTXT; CNKI; VEN; WOTXT; USTXT; EPTXT; NCBI; GenBank: ASM, SSL BH, ft, SEQ ID NO: 1-10, Cas9. epilepsy, model, PKHD1, primer C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. PX CN 116218905 A (HUASHAN HOSPITAL, FUDAN UNIVERSITY) 06 June 2023 (2023-06-06) claims 1-10, and description, paragraphs [0003]-[0097] 1-15 A A CN 113897399 A (ZHEJIANG SAIWEISI BIOTECHNOLOGY CO., LTD.) 07 January 2022 (2022-01-07) description, paragraphs [0004]-[0066], and figures 1-7 CN 109820845 A (CHINA MEDICAL UNIVERSITY) 31 May 2019 (2019-05-31) description, paragraphs [0005]-[0039J, and figures 1-6 1-22 1-22 A CN 111004818 A (NANJING MATERNITY AND CHILD HEALTH HOSPITAL et al.) 14 April 2020 (2020-04-14) entire document 1-22 A CN 101485917 A (SHENZHEN INSTITUTE OF ADVANCED TECHNOLOGY) 22 July 2009 (20)9-07-22) entire document 1-22 | | Further documents are listed in the continuation of Box C. | J | See patent family annex. * Special categories of cited documents: “T” later document published after the international filing date or priority “A” document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand the to be of particular relevance principle or theory underlying the invention “D” document cited by the applicant in die international application “X” document of particular relevance; the claimed invention cannot be “E" earlier application orpatent but published on or after the international considered novel or cannot be considered to involve an inventive step filing date when the document is taken alone •SL” document which may throw doubts on priority claim(s) or which is “Y” document of particular relevance; the claimed invention cannot be cited to establish the publication date of another citation or other considered to involve an inventive step when the document is special reason (as specified) combined with one or more other such documents, such combination “O” document referring to an oral disclosure, use, exhibition or other being obvious to a person skilled in the art means document member of the same patent family “P” document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search 20 November 2023 Date of mailing of the international search report 01 December 2023 Name and mailing address of the ISA / CN China National Intellectual Property Administration (ISA / CN) China No. 6, Xitucheng Road, Jimenqiao, Haidian District, Beijing 100088 Authorized officer Telephone No.INTERNATIONAL SEARCH REPORT International application No. PCT / CN2023 / 120325C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. A CN 112168420 A (HUAZHONG UNIVERSITY OF SCIENCE AND TECHNOLOGY) 05 January 2021 (2021-01-05) entire document 1-22INTERNATIONAL SEARCH REPORT International application No. PCT / CN2023 / 120325 Box No. I Nucleotide and / or amino acid sequence(s) (Continuation of item l.c of the first sheet)1. With regard to any nucleotide and / or amino acid sequence disclosed in the international application, the international search was carried out on the basis of a sequence listing: a. / forming part of the international application as filed. b. __ furnished subsequent to the international filing date for the purposes of international search (Rule 13ter. 1(a)),accompanied by a statement to the effect that the sequence listing does not go beyond the disclosure in the international application as filed.

2. | | With regard to any nucleotide and / or amino acid sequence disclosed in the international application, this report has been established to the extent that a meaningful search could be carried out without a WIPO Standard ST.26 compliant sequence listing.

3. Additional comments:INTERNATIONAL SEARCH REPORT Information on patent family membersInternational application No.PCT / CN2023 / 120325Patent document cited in search report Publication date (day / month / year) Patent family member)s) Publication date (day / month / year) CN 116218905 A 06 June 2023 None CN 113897399 A 07 January 2022 None CN 109820845 A 31 May 2019 None CN 111004818 A 14 April 2020 None CN 101485917 A 22 July 2009 CN 101485917 B 05 January 2011 CN 112168420 A 05 January 2021 None

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