Construction and application of a non-human animal model of Cri du Chat syndrome gene knockout

Abstract

The present invention discloses a method for constructing and applying a gene knockout non-human animal model. The model is constructed based on CRISPR / Cas9 gene knockout technology. The construction method comprises the following steps: Step 1: Designing sgRNA and Cas9 RNA, transcribing them into mRNA in vitro, and microinjecting the active sgRNA and Cas9 RNA into fertilized eggs of non-human animals to obtain a gene knockout non-human animal; Step 2: Identifying the gene knockout non-human animal model. The advantages of this method are as follows: Using CRISPR / Cas9 gene knockout technology, the present invention establishes the first gene knockout non-human animal model for Cry Me a Cat syndrome, providing a convenient and reliable animal model for studying and screening drugs for treating Cry Me a Cat syndrome.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for constructing a gene knockout non-human animal model and its application based on CRISPR / Cas9 technology. Background Art

[0002] Cri Du Chat syndrome is a hereditary neurodevelopmental disorder caused by chromosomal structural abnormalities due to partial or complete deletion of the short arm of chromosome 5 (5pmicrodeletion, 5p deletion). It is also known as 5p deletion syndrome or pediatric Cri Du Chat syndrome. The size of the deletion of the short arm of chromosome 5 varies among different individuals with Cri Du Chat syndrome (referred to as 5p patients), ranging from 0.5Mb to 45Mb (the deletion position is from the 5p15 region to the entire short arm).

[0003] Internationally, Nguyen et al. (2015) identified 11 genes within the 5p arm that are associated with Cry-à-l'-cat syndrome. Haploinsufficiency of five genes, TERT, SEMA5A, MARCHF6, CTNND2, and NPR3, can directly cause Cry-à-l'cat syndrome. Haploinsufficiency of the remaining six genes, SLC6A3, CDH18, CDH12, CDH10, CDH9, and CDH6, combined with environmental factors, can also lead to Cry-à-l'-cat syndrome. Furthermore, haploinsufficiency of RICTOR and DAB2 can also cause Cry-à-l'-cat syndrome. Nevado et al. (2021) used high-resolution single nucleotide polymorphism (SNP) arrays, cytogenetics, in situ fluorescence hybridization (FISH), and multiplex ligation-linked probe amplification (MLPA) technologies to conduct a molecular diagnostic study on a cohort of 70 unrelated children with 5p, with an average age of 9 years. They determined the size, range, gene content, and genetic information of gene rearrangements of the 5p defect in most children, and established a genotype-clinical phenotype relationship for children with 5p based on clinical and molecular data. Combining data from another 7 research groups, through a meta-analysis of 423 children with 5p, Nevado et al. found that approximately 95%-99% of children had developmental delay and hypotonia, approximately 80% had violent aggressive behavior and other behavioral abnormalities, 65% had microcephaly and intellectual disability, 42% had clinical manifestations of autism, and 33%-36% had cardiac and gastrointestinal disorders. In addition, about 95% of 5p girls have epilepsy, while less than 6% of 5p boys have epilepsy, the cause of which is unknown.

[0004] Existing methods for studying Cri du Chat syndrome (Cri du Chat syndrome) primarily rely on clinical or epidemiological studies. Cohort studies, a type of epidemiological research approach, select two groups of people, either exposed or not, or stratify them into subgroups based on exposure level, track their respective outcomes (morbidity and mortality), and compare the differences in outcomes between the two groups or within each group to determine the presence of a causal relationship between the exposure factor and the onset of the disease, as well as the extent of the association. However, methods for constructing gene knockout animal models for Cri du Chat syndrome have not yet been reported. Summary of the Invention

[0005] The non-human animal model of cat cry syndrome of the present invention has some unique advantages: experimental conditions are easy to control; non-human animals reproduce quickly, which is shorter than clinical research time; and breeding of non-human animals is more economical in terms of housing, equipment, feed, management, etc.

[0006] The Cry-a-cat syndrome model not only validates the key gene for Cry-a-cat syndrome but also lays a solid foundation for in-depth research on this rare pediatric neurological disorder. Compared to existing clinical and epidemiological studies, it allows for deeper mechanistic insights, better control of experimental conditions, and is more time-efficient than clinical research.

[0007] The present invention provides a method for constructing a gene knockout non-human animal model, characterized in that the construction method comprises the following steps:

[0008] S1, determine the specific target site sgRNA1 and sgRNA2, and transcribe them into mRNA in vitro with Cas9 nuclease;

[0009] S2, microinjecting active sgRNA1, sgRNA2, and Cas9 RNA into non-human mammalian fertilized eggs to obtain gene knockout non-human animals;

[0010] The sgRNA1 is selected from the group consisting of SEQ ID NOs: 5-12, and the sgRNA2 is selected from the group consisting of SEQ ID NOs: 14-26; preferably, the sgRNA1 is shown as SEQ ID NO: 7, and the sgRNA2 is shown as SEQ ID NO: 24.

[0011] In some embodiments, step S2 comprises the following steps:

[0012] S21, ovulation induction and in vitro fertilization of non-human animals, and cultivation of fertilized eggs;

[0013] S22, microinjection of active sgRNA1, sgRNA2, and Cas9 RNA into non-human animal fertilized eggs;

[0014] S23, in vitro culture of fertilized eggs, implantation into recipients and cultivation of targeted gene-modified animals.

[0015] In some embodiments, step S23 comprises the following steps:

[0016] S231, taking the fertilized egg that survives the injection and transplanting it into a mature female non-human animal to produce a non-human animal, which is the F0 generation non-human animal;

[0017] S232, extract tail DNA from F0 non-human animals, amplify by PCR, and send the product for sequencing;

[0018] S233, mating the positive F0 generation non-human animals with wild-type non-human animals of the opposite sex to obtain F1 generation heterozygous non-human animals;

[0019] S234, hybridizing F1 generation heterozygous non-human animals to obtain F2 generation homozygous non-human animals, which are non-human animal models.

[0020] In some embodiments, approximately 1.68 Mb of bases are deleted in the positive F0 generation non-human animal.

[0021] In some embodiments, the base sequence deleted in the positive F0 generation non-human animal is a DNA fragment from position 83120000 to position 84800000 of the chr2 gene.

[0022] The non-human animal is a non-human mammal, preferably a rat. Therefore, the present invention further discloses a method for constructing a gene knockout rat model, characterized in that the construction method comprises the following steps:

[0023] S1, determine the specific target site sgRNA1 and sgRNA2, and transcribe them into mRNA in vitro with Cas9 nuclease;

[0024] S2, active sgRNA1, sgRNA2, and Cas9 RNA were microinjected into rat fertilized eggs to obtain gene knockout rats;

[0025] The sgRNA1 is selected from the group consisting of SEQ ID NOs: 5-12, and the sgRNA2 is selected from the group consisting of SEQ ID NOs: 14-26; preferably, the sgRNA1 is shown as SEQ ID NO: 7, and the sgRNA2 is shown as SEQ ID NO: 24.

[0026] In some embodiments, step S2 comprises the following steps:

[0027] S21, rat ovulation induction and in vitro fertilization, cultivation of fertilized eggs;

[0028] S22, active sgRNA1, sgRNA2, and Cas9 RNA were microinjected into rat fertilized eggs;

[0029] S23, in vitro culture of fertilized eggs, implantation into recipients and cultivation of targeted gene-modified animals.

[0030] In some embodiments, step S23 comprises the following steps:

[0031] S231, the fertilized eggs that survived the injection were transplanted into mature female rats to produce rats, which are the F0 generation rats;

[0032] S232, extract tail DNA from F0 generation rats, amplify by PCR and send the product for sequencing;

[0033] S233, the positive F0 generation rats were mated with wild-type rats of the opposite sex to obtain F1 generation heterozygous rats;

[0034] S234, hybridizing F1 generation heterozygous rats to obtain F2 generation homozygous rats, which are non-human animal models.

[0035] In some embodiments, approximately 1.68 Mb of bases are deleted in the positive F0 generation rat.

[0036] In some embodiments, the base sequence deleted in the positive F0 generation rat is a DNA fragment from position 83120000 to position 84800000 of the chr2 gene.

[0037] On the other hand, the present invention provides a gene targeting vector, characterized in that the vector is an sgRNA expression vector based on the CRISPR / Cas9 system, wherein sgRNA1 is selected from the sequence of the group consisting of SEQ ID NO: 5-12, and sgRNA2 is selected from the sequence of the group consisting of SEQ ID NO: 14-26; preferably, the sgRNA1 is as shown in SEQ ID NO: 7, and the sgRNA2 is as shown in SEQ ID NO: 24.

[0038] On the other hand, the present invention provides a use of the sgRNA according to claim 8 in constructing a gene knockout non-human animal model related to cat cry syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The present invention can be more fully understood with reference to the following drawings.

[0040] Figure 1 The 5p model rat - rat chromosome 2 knockout range is shown.

[0041] Figure 2 Shown is a schematic diagram of the preparation of 5p model rats.

[0042] Figure 3 A map of the pre-cut pCS vector is shown.

[0043] Figure 4 The results of sgRNA activity detection are shown.

[0044] Figure 5 The figure shows the RNA electropherogram during the RNA preparation of sgRNA.

[0045] Figure 6 The primer design principles for identifying primers in the genotyping of F0 generation rats are shown.

[0046] Figure 7 The figure shows the genotype identification of the F0 generation rat tail, and the identification results are obtained using primers EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-Mut-R.

[0047] Figure 8 The primer design principles for identifying primers in F1 generation rat genotype identification are shown.

[0048] Figure 9 The figure shows the identification results of the tail genotype of F1 rats using primers EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-WT-R.

[0049] Figure 10 The figure shows the identification results of the tail genotype of F1 rats using the primers EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-Mut-R.

[0050] Figure 11 Key homologous genes in the 5p model rat are shown.

[0051] Figure 12 It was shown that the 5p model rats had growth retardation.

[0052] Figure 13 The results showed that the motor coordination ability of 5p model rats was delayed.

[0053] Figure 14 The 5p model rats showed hypotonia.

[0054] Figure 15 The results showed that 5p model rats had obvious social impairment.

[0055] Figure 16 It was shown that 5p model rats have repetitive stereotyped behaviors and lack of curiosity.

[0056] Figure 17It was shown that 5p model rats exhibited obvious anxiety-like behaviors.

[0057] Figure 18 The results showed that the limb balance ability of 5p model rats decreased.

[0058] Figure 19 It shows that 5p model rats have cognitive impairment.

[0059] Figure 20 The results showed that the activity of 5p model rats decreased, accompanied by obvious anxiety-like behaviors. Detailed description

[0060] Various features and aspects of the present invention are discussed in greater detail below. Example

[0061] The following examples are illustrative only and are not intended to limit the scope or content of the invention in any way.

[0062] Example 1: Sequencing confirmation of target sequence

[0063] The target gene sequence may vary between strains. To ensure the efficiency of the designed Cas9 / sgRNA, the target site sequence of the SD rat tail must first be PCR amplified and sequenced to ensure that the sgRNA recognition sequence is completely consistent with the SD rat tail DNA sequence. The PCR primers are as follows:

[0064] Table 1. Primer sequence list

[0065]

[0066] PCR and sequencing were performed on SD rat tail DNA, and the results showed that the SD rat tail target sequence was completely consistent with the sequences given in Genebank and Ensembl.

[0067] Example 2: Cas9 / sgRNA design and construction

[0068] 1. Cas9 / sgRNA design

[0069] Based on the design principles of sgRNA, 8 sgRNAs were designed in the 5' target site and 14 sgRNAs were designed in the 3' target site region. The corresponding targeting sequences are as follows:

[0070] Table 2. Targeting sequence information corresponding to sgRNA

[0071]

[0072]

[0073] 2. Construction of Cas9 / sgRNA Plasmid

[0074] Oligos were synthesized according to the designed sgRNA sequence and connected to the pCS-4G vector by Gibson method. The ligation product was transformed and sent for sequencing to verify correctness. Figure 3 shown.

[0075] A rat gene homologous to the human 5p gene, located on rat chromosome 2, with a 1.68 Mb deletion (chr2:83120000-84800000);

[0076] sgRNAs were designed in the non-conserved regions upstream of chr2:83120000 and downstream of chr2:84800000, respectively, resulting in a gene deletion of approximately 1.68 Mb, thereby achieving the desired gene knockout. The rat model was established using the EGE system developed by Biocytogen based on CRISPR / Cas9.

[0077] Example 3: Activity detection of Cas9 / sgRNA

[0078] The activity of sgRNA was detected using a CRISPR / Cas9 activity detection method independently developed by Biocytogen, the UCATM method, which has the advantages of no species restriction, high throughput, wide adaptability, high sensitivity, and simplicity. EGE-ZLM-048-A-sgRNA3 and EGE-ZLM-048-A-sgRNA20 were selected for the next experiment. The test results are as follows Figure 4 shown.

[0079] Example 4: RNA preparation of sgRNA

[0080] EGE-ZLM-048-A-sgRNA3 and EGE-ZLM-048-A-sgRNA20 were transcribed in vitro to obtain RNA for microinjection. The RNA electrophoresis diagram is shown in Figure 2. Figure 5 shown.

[0081] Example 5: Microinjection of Cas9 / sgRNA

[0082] Cas9 / sgRNA was microinjected into rat fertilized eggs, and the birth conditions of F0 rats after injection were as follows.

[0083] Table 3. F0 rat births after Cas9 / sgRNA injection into rat fertilized eggs

[0084]

[0085] Example 6 Identification of F0 Generation Rat Genotype

[0086] Gene knockout rats were constructed by injecting fertilized eggs with Cas9 / sgRNA. Due to the rapid cleavage rate of early embryos, the resulting F0 rats are chimeric. Therefore, the F0 genotype obtained by tail analysis of the F0 rats is for reference only and does not guarantee a heritable gene mutation. Heritable genotypes must be confirmed by tail analysis of the F1 rats.

[0087] 1. Identification Primer Design

[0088] Primer design principles such as Figure 6 shown.

[0089] Primer information is as follows:

[0090] Table 4. Identification primer information

[0091]

[0092] PCR conditions: 2x Taq Plus Master Mix II (Dye Plus) progress

[0093] Enzyme: 2x Taq Plus Master Mix II (Dye Plus)

[0094] Table 5. PCR reaction conditions and procedures

[0095]

[0096] 2. Identification of F0 generation rat tail genotype

[0097] Identification results such as Figure 7 Primers: EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-Mut-R.

[0098] PCR amplification and product sequencing showed that EM48-0002, EM48-0004, EM48-0005, EM48-0007, EM48-0008, EM48-0019, EM48-0028, EM48-0032, EM48-0045 and EM48-0052 were positive F0 rats.

[0099] Example 7 Identification of F1 Generation Rat Genotype

[0100] Some of the F0 generation rats identified as positive were mated with wild-type rats to obtain F1 generation rats with stable genotypes. The mating results are as follows:

[0101] Table 6. Mating of F0 generation rats and wild type rats

[0102]

[0103] 1. Identification Primer Design

[0104] Primer design principles such as Figure 8 shown.

[0105] Primer information is as follows:

[0106] Table 7. Identification primer sequence information

[0107]

[0108] Polymerase chain reaction conditions: 2x Taq Plus Master Mix II (Dye Plus) progress

[0109] Enzyme: 2x Taq Plus Master Mix II (Dye Plus)

[0110] Table 8. PCR reaction conditions and procedures

[0111]

[0112] The genotype of the F1 generation rat tail was identified. The identification results were as follows: Figure 9 As shown, primers: EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-WT-R; Figure 10 As shown, primers: EGE-ZLM-048-A-WT-F / EGE-ZLM-048-A-Mut-R.

[0113] F1 generation sequencing results:

[0114] 1EM48-0001, 1EM48-0004, 1EM48-0005, 1EM48-0007, 1EM48-0009, 1EM48-0010, 1EM48-0011, 1EM48-0012, 1EM48-0013, 1EM48-0015, 1EM48-0016 and 1EM48-0020 are positive F1 rats.

[0115] We used positive rats for subsequent experiments.

[0116] The rat gene homologous to the human 5p gene is located on rat chromosome 2. The 1.68Mb knockout fragment contains multiple genes related to growth and development. Preliminary results show that homozygous 5p model mice die in utero, while heterozygous 5p model mice can survive, have slowed growth and development, and are significantly lower in weight and body length than normal rats. Compared with control rats of the same age, the 5p model mice have low muscle tone. These phenotypes are highly consistent with those of 5p patients.

[0117] Figure 11 qPCR experiments showed that 5p model rats lacked the key pathogenic homologous genes of cat cry syndrome, including Ctnnd2, Cct5, Marchf6, and Ankrd33b.

[0118] Figure 12 AC showed that the weight, body length and tail length of 5p model rats were significantly smaller than those of control rats. Figure 12 DE showed that there was no significant change in the auricle development of 5p model rats and the time of first eye opening of suckling rats compared with control rats.

[0119] Interestingly, compared with control rats, 5p model rats showed a significantly earlier first postnatal reactivity to environmental sounds, indicating hypersensitivity to ambient sounds in 5p model rats, a phenotype highly consistent with 5p patients. Similar to Ctnnd2 knockout mice, we assessed the animals' social behavior. Results from a three-chamber social behavior test revealed severe social impairment in 5p model rats. Similar to patients with Cry-a-Cat syndrome, 5p model rats displayed significantly increased fur-stroking time and repetitive, stereotyped behaviors, accompanied by decreased curiosity about their surroundings, suggesting typical autistic-like behaviors. An open-field test revealed a significant decrease in the range of activity in 5p model rats, with activity concentrated primarily in the periphery of the open field, and significantly less time spent in the central area, indicating significant anxiety-like behaviors. An elevated maze test also confirmed significant anxiety-like behaviors in 5p model rats. We also assessed the animals' limb balance and postural gait, revealing a degree of impairment in motor balance and coordination in 5p model rats, suggesting abnormalities in cerebellum and extrapyramidal system function. Further behavioral experiments showed that the 5p model rats had obvious deficits in novel object preference memory, object location recognition memory, and object location exchange recognition cognitive functions, which were highly consistent with the intellectual disability of patients with cat cry syndrome.

[0120] Figure 13 AB showed that the development of plane and aerial righting reflex in 5p model rats was delayed; Figure 13 C shows that the time of the first rotation movement after birth of 5p model rats is slightly delayed, but there is no significant difference compared with the control rats. Figure 13 D shows that compared with the control mice, the time of first crawling and negative geotaxis in 5p model mice did not change significantly. Figure 13 E Compared with the control rats, the first reaction of the 5p model rats to environmental sounds after birth was significantly earlier, indicating that the 5p model rats have hypersensitivity to environmental sounds.

[0121] Figure 14 AB showed that compared with the control normal rats, the total time and score of the 5p model rats in grasping the rope were significantly decreased, indicating that the muscle tension of the 5p model rats was reduced. Figure 15It showed that 5p model rats had obvious social impairment.

[0122] Figure 16 A shows that the hair-stroking time of 5p model rats was significantly increased, suggesting that the 5p model rats have repetitive stereotyped behaviors. Figure 16 B shows that compared with the control rats, the digging time of the 5P model rats in the cage was significantly reduced, indicating that the 5P model rats lack curiosity about the surrounding things.

[0123] Figure 17 A, Open field test: The 5p model rats spent significantly less time in the central area of ​​the open field, showing obvious anxiety-like behavior, but the animal's movement speed in the open field did not change. Figure 17 B shows that the 5p model rats also exhibited obvious anxiety-like behaviors in the elevated plus maze test, further supporting the results of the open field test.

[0124] Figure 18 A shows abnormalities in the limb balance ability test of 5p model rats. Figure 18 B shows that the movement posture and gait of the 5p model rats are basically normal. The results show that there is no obvious loss of movement coordination in the 5p model rats, suggesting that the cerebellum and extrapyramidal system functions of the 5p model rats are abnormal to a certain extent.

[0125] Figure 19 AC showed that the 5p model rats had obvious deficits in novel object preference memory, object location recognition memory, and object location exchange recognition cognitive functions.

[0126] In summary, the phenotype of these 5p model rats is highly consistent with that of individuals with Cri du Chat syndrome. Our establishment of this first rodent model not only validates the key gene for Cri du Chat syndrome, but also lays a solid foundation for in-depth research into the pathogenesis of this rare pediatric neurological disease and for drug development.

[0127] Incorporated by Reference

[0128] Each patent and scientific document mentioned herein is incorporated by reference in its entirety for all purposes.

[0129] Equivalence

[0130] The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered in all cases as illustrative rather than limiting of the invention described herein. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. A method for constructing a gene knockout rat model related to cat cry syndrome, characterized in that: The construction method comprises the following steps: S1, determine the specific target site sgRNA1 and sgRNA2, and transcribe them into mRNA in vitro with Cas9 nuclease; S2, rat ovulation and in vitro fertilization, culture of fertilized eggs, microinjection of active sgRNA1, sgRNA2 and Cas9 RNA into rat fertilized eggs, and culture of fertilized eggs in vitro; S3, the fertilized eggs that survived the injection were transplanted into mature female rats to produce rats, which are the F0 generation rats; S4, extract tail DNA from F0 generation rats, amplify by PCR and send the product for sequencing; S5, the positive F0 generation rats were mated with wild-type rats of the opposite sex to obtain F1 generation heterozygous rats; this is the rat model, The sgRNA1 is shown in SEQ ID NO: 7, and the sgRNA2 is shown in SEQ ID NO:

24. The base sequence deleted in the positive F0 generation rats is a DNA fragment in the region from position 83120000 to position 84800000 of the chr2 gene.

2. A gene targeting vector related to cat cry syndrome, characterized in that: The vector is an sgRNA expression vector based on the CRISPR / Cas9 system, comprising sgRNA1 and sgRNA2, wherein the sgRNA1 is shown as SEQ ID NO: 7, and the sgRNA2 is shown as SEQ ID NO:

24.

3. Use of sgRNA in constructing a gene knockout rat model associated with cat cry syndrome, wherein the sgRNAs are sgRNA1 and sgRNA2, wherein the sgRNA1 is shown in SEQ ID NO: 7, and the sgRNA2 is shown in SEQ ID NO: 24.

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