Targeted macaque ATM gene knockout sgRNA and application

Abstract

The invention discloses sgRNA for targeted macaque ATM gene knockout and application, and belongs to the technical field of biology. The sgRNA nucleotide sequence of the targeted Macaca mulatta ATM gene provided by the invention is shown as any one of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. The method comprises the following steps: designing a plurality of sgRNAs aiming at the ATM gene of the macaque on the basis of a CRISPR / Cas9 system, jointly injecting the sgRNA for targeted macaque ATM gene knockout and Cas9 protein or a mixture of the sgRNA for targeted macaque ATM gene knockout and Cas9 mRNA into a macaque embryo, and then transplanting the embryo into a receptor female macaque body to produce the macaque with the ATM gene knockout. The method provides a new method for establishing a non-human primate AT animal model, and provides a research basis for researching the cerebellum degenerative phenotype, pathological characteristics and internal molecular mechanism of AT patients, analyzing the pathogenic mechanism and screening drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to targeting macaques ATM sgRNA for gene knockout and its applications. Background Art

[0002] Ataxia-telangiectasia (AT) is a rare autosomal recessive genetic disease caused by the loss of ATM function, characterized by cerebellar degeneration, extrapyramidal reactions, oculomotor disorders, telangiectasia, immune dysfunction, endocrine disorders, growth retardation, cancer susceptibility and radiation sensitivity. ATM (Ataxia telangiectasia mutated) belongs to the phosphatidylinositol 3-kinase related kinase family (PI3KK), has 64 exons and a molecular weight of 370kD. ATM protein is a serine-threonine protein kinase associated with DNA damage response and cell cycle regulation. It can phosphorylate more than 700 substrates and is very important for accurately and effectively maintaining the genome integrity of living organisms. Some AT patients develop severe ataxia and cerebellar atrophy in the early stages, and after the age of ten, eye coordination is impaired, making reading increasingly difficult, accompanied by fine motor dysfunction, such as difficulty writing, coloring, and eating with tableware, as well as symptoms such as easy fatigue and difficulty concentrating.

[0003] Although the prior art ATM Knockout mice show a variety of symptoms similar to those of human AT patients, including growth retardation, meiotic defects, immune abnormalities, radiation sensitivity, and cancer susceptibility. However, current rodent models are defective, lacking the core ataxia symptoms of AT and severe neurodegeneration, such as neurodegeneration or lack of stable motor dysfunction, and cannot simulate the key disease phenotypes of AT patients. Therefore, there is an urgent need to develop more suitable animal models that will help observe the entire process of disease occurrence and development and promote the understanding of the pathogenesis of AT; at the same time, it will also provide an ideal animal model for finding available drug treatment targets.

[0004] In recent years, the CRISPR / Cas9 system has rapidly become a leader in gene editing tools due to its high efficiency, simple operation, and low cost, and has been widely used in gene function research, disease models, gene therapy, and other fields. ATM Gene reports have not targeted macaques ATM Reports of sgRNA for gene knockout. Summary of the invention

[0005] In view of the above technical problems, the present invention intends to provide a method targeting macaques ATM sgRNA for gene knockout and its application. The technical solution of the present invention is as follows: The present invention provides a method for targeting macaques ATM The sgRNA for gene knockout, wherein the nucleotide sequence of the sgRNA is at least one of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

[0006] Preferably, the nucleotide sequence of the sgRNA is a combination of SEQ ID NO: 2 and SEQ ID NO: 6 or a combination of SEQ ID NO: 7 and SEQ ID NO: 8.

[0007] The present invention also protects the sgRNA in targeting macaques ATM Application in gene knockout.

[0008] The present invention also protects a macaque ATM The method for constructing a gene knockout model comprises the following steps: S1 will target macaques ATM Gene knockout sgRNA and Cas9 protein may target macaques ATM The knockout sgRNA and Cas9 mRNA mixture were co-injected into macaque embryos to obtain ATM Knockout macaque embryos; After in vitro screening, S2 was injected into the cells obtained from S1 by microinjection. ATM The knockout macaque embryos were transplanted into recipient female monkeys to produce ATM Knockout macaques.

[0009] The present invention also protects macaques ATM The application of gene knockout models in studying the occurrence and development mechanism of AT or screening therapeutic drugs for AT.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The present invention designs and screens ATM CRISPR / Cas9 knockout system for genes, providing targeted ATM The sgRNA for gene knockout was successfully constructed ATM Gene knockout macaques. Phenotypic analysis revealed: ATMThe knockout macaques showed typical AT symptoms, such as cerebellar atrophy, movement disorders, conjunctival telangiectasia, growth retardation, immunosuppression, and abnormally elevated alpha-fetoprotein levels, which were consistent with those of AT patients, and better simulated the disease phenotype of early AT in humans. Among them, some core phenotypes did not appear in rodent models, such as cerebellar vermis, hemispheric atrophy, and ataxia symptoms, or phenotypes that were difficult to observe, such as telangiectasia and fine motor movements. The method of the present invention is for studying ATM Gene-related diseases such as AT-related diseases provide a method to construct animal models, providing a research basis for understanding their pathogenic mechanisms and drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 are the cutting effects of two groups of sgRNA, Figure 1 A is the cutting effect of combination 1 (sgRNA2+sgRNA6), Figure 1 B shows the cutting effect of combination 2 (sgRNA7+sgRNA8).

[0012] Figure 2 for ATM The genotype and ATM protein expression of knockout macaques, Figure 2 A shows the target gene mutation in the AT group; Figure 2 B is ATM Figure 1 shows the ATM-mRNA detection in knockout macaques; Figure 2 C shows the expression of ATM protein in AT group fibroblasts.

[0013] Figure 3 This is a comparison of the conjunctival capillary dilation in macaques.

[0014] Figure 4 This is a comparison of the physical development of the WT group and the AT group. Figure 4 A is a comparison of body weight between the WT group and the AT group, *: P < 0.05; Figure 4 B is the head circumference comparison between WT group and AT group, *: P<0.05, Figure 4 C is a comparison of the double diameter tops between the WT group and the AT group *: P < 0.05; Figure 4 D is the comparison of body length between the WT group and the AT group, **: P < 0.01, paired t-test (two-tailed), mean ± SEM.

[0015] Figure 5 This is a comparison chart of chest circumference and abdominal circumference between the WT group and the AT group. Figure 5 A is the comparison of abdominal circumference between WT group and AT group. Figure 5 B is the comparison of abdominal circumference between WT group and AT group. Paired t-test (two-tailed), mean ± SEM.

[0016] Figure 6 This is a comparison of the upper limbs between the WT group and the AT group. Figure 6 AD are the comparison of arm length, palm length, upper arm circumference and lower arm circumference of AT and WT groups, respectively. Paired t-test (two-tailed), at 9 months of age, Ratio paired t-test correction, mean ± SEM, ***: P < 0.001, *: P < 0.05.

[0017] Figure 7 This is a comparison of the lower limbs between the WT group and the AT group. Figure 7 AD are comparisons of leg length, foot length, thigh circumference and calf circumference between the WT group and the AT group, **: P<0.01.

[0018] Figure 8 This is a comparison of blood analysis of macaques in the WT group and the AT group. Figure 8 A is a comparison of the white blood cell levels between the WT group and the AT group, *: P < 0.05; Figure 8 B is a comparison of lymphocyte levels between the WT group and the AT group, ***: P < 0.001, *: P < 0.05; Figure 8 C is a comparison of hemoglobin levels between the WT group and the AT group, *: P < 0.05; Figure 8 D is a comparison of immunoglobulin IgG levels between the WT group and the AT group, *: P < 0.05, corrected by Ratio paired t-test at 6 months, paired t-test (two-tailed), mean ± SEM.

[0019] Fig. 9 This is a comparative analysis of the immune levels of macaques in the WT group and the AT group. Fig. 9 A is a comparison of immunoglobulin IgM levels between the WT group and the AT group, paired t-test (two-tailed), mean ± SEM, *: P < 0.05; Fig. 9 B is a comparison of alpha-fetoprotein levels between the WT group and the AT group, paired t-test (two-tailed), mean ± SEM, *: P < 0.05, because the values ​​of the WT group at 12 and 15 months were too small, the differences between the two groups were not equal, and the Ratio paired t-test was used to correct it.

[0020] Fig.10 Comparison of sagittal sections of the brains of AT and WT monkeys at 6 and 12 months of age. The left image shows the comparison of the cerebellar hemispheres of the macaques, and the right image is the comparison of the largest cross-sectional images, showing the comparison of the cerebellar vermis.

[0021] Fig.11 is a comparison chart of gait cycle, stride, frequency, and step length, where Fig.11 A is a statistical comparison of gait cycles between WT and AT groups. Fig.11 B is a comparison chart of step frequency statistics. Fig.11 C is a stride comparison chart. Fig.11D is the step length comparison chart, Fig.11 AC: Mann-Whitney rank sum test (two-tailed); Fig.11 D: Welch's test correction (two-tailed), all expressed as mean ± SEM, **: P < 0.01, *: P < 0.05.

[0022] Fig.12 Comparison of step interval and dynamic gait of macaques in the WT group and AT group. Fig.12 A is a statistical comparison of the step intervals of macaques in the WT group and the AT group. Fig.12 B is the support comparison diagram. Fig.12 C is the swing phase contrast diagram, Fig.12 D is a comparison diagram of standing on both feet. Fig.12 A: Welch's test correction (two-tailed), Fig.12 BD: unpaired t-test (two-tailed), mean ± SEM, ****: P < 0.0001, **: P < 0.01, *: P < 0.05. DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0024] Experimental animals and reagents: The macaque donors and recipients used in this experiment were from the Kunming Primate Center (KPRC) of the Chinese Academy of Sciences. They were fed according to standard procedures, and all embryos in the experiment came from KPRC. The Cos7 cell line (African green monkey kidney cell) samples used in the experiment came from the Kunming Cell Bank of the Chinese Academy of Sciences. The vector selection Cos7 cell line plasmid system: pSpCas9(BB)-2A-GFP.

[0025] Example 1 Targeting macaques based on the CRISPR / Cas9 system ATM Design and screening of sgRNA for gene knockout Analysis of macaques by bioinformatics ATM Gene sequence, and combined with mature CRISPR target point detection software to design specific sgRNAs. First, the Cos7 cell line was used for preliminary screening in vitro, and the screened sgRNA was further verified in fertilized eggs, and transplanted after the knockout efficiency was determined.

[0026] 1. sgRNA design For macaques ATM Several major exons were designed with multiple specific sgRNAs, and NCBI searched for macaque ( Macaca mulatta )of ATM Gene ID: 100427400, obtained from macaque ATM Gene information, a total of 64 exons. Find the PAM sequence in the target gene, and preferably select sgRNA with a difference of more than 3 bases between the target and non-target genes, as close to the PAM as possible. Target online analysis tools and off-target prediction software: http: / / crispr.mit.edu, http: / / chopchop.cbu.uib.no, http: / / crispor.tefor.net, http: / / www.rgenome.net / cas-offinder / , sgRNA sequence targeting the ATM gene. The designed 8 target sequences are shown in Table 1 below, and their nucleotide sequences are shown in SEQ ID NO: 1-8 respectively: Table 1 sgRNA sequence list

[0027] 2. Knockout based on CRISPR / Cas9 system ATM Screening of sgRNA for genes 2.1 In vitro transcription and synthesis of Cas9 mRNA and sgRNA Cas9 mRNA and sgRNA were obtained by in vitro transcription using the T7 in vitro transcription kit. When preparing Cas9 mRNA and sgRNAs, the T7 promoter sequence was added to the Cas9 or sgRNAs template by PCR amplification. The T7-Cas9 / sgRNA PCR product was gel purified and in vitro transcribed using a commercial kit, the T7 in vitro transcription kit (Thermo, K0441). Cas9 mRNA and sgRNA were purified using the MinElute PCR purification kit. After quality inspection, the final concentration of the obtained Cas9 mRNA was 200 ng / μL and stored at -80°C, and the final concentration of the obtained sgRNA was 20 ng / μL and stored at -80°C.

[0028] 2.2 Detection of knockout efficiency of sgRNA 1-6 in Cos7 cells The plasmid vector (pSpCas9 (BB) -2A-GFP) of sgRNA 1-6 was constructed and transiently transfected into Cos7 cells, and the editing efficiency was detected by TA cloning. The results are shown in Table 2.

[0029] Table 2 Comparison of knockout efficiency of sgRNA 1-6 in Cos7 cells

[0030] From Table 2 , we can see the knockout efficiency of sgRNA1-6 in Cos7 cells. The detection number is the randomly selected clones for sequencing. The transfection efficiency is roughly estimated by comparing the transfection-positive cells. The mutation rate is (mutation number / detection number) / transfection efficiency. It can be seen that sgRNA2, sgRNA4 and sgRNA6 have high in vitro efficiencies, all exceeding 50%.

[0031] 2.3 Knockout efficiency of sgRNAs in embryos 2.3.1 Knockout efficiency of sgRNA2 and sgRNA4 in embryos The mixed solutions of sgRNA2, sgRNA4 and Cas9 mRNA obtained above were injected into macaque embryos respectively using embryo micromanipulation technology, and the embryonic development after injection was observed and the knockout efficiency was detected. During injection, Cas9 mRNA was fully mixed with the two sgRNAs, and the concentration was controlled with nuclease-free water. Specifically: Cas9 mRNA / sgRNA2 (No. 1-10) or sgRNA4 (No. 11-16) (where the concentration of Cas9 mRNA was 100ng / μL, and the concentrations of sgRNA2 and sgRNA4 were 10ng / μL). Injected into 16 fertilized eggs in good condition, including 11 in the MII stage, 0 in the MI stage, and 5 in the GV stage, cultured in vitro for one week. In addition, 2 blank fertilized eggs were selected as negative controls.

[0032] The results are shown in Table 3. Among the 10 fertilized eggs injected with sgRNA2 (No. 1-10), 6 developed to the morula stage, 4 even developed to the blastocyst stage, and 4 developed to the 8-16 cell stage. Cloning and sequencing found that gene mutations related to the sgRNA2 target sequence were detected in samples from 9 fertilized eggs, including base deletions and insertions, and the positive embryo knockout efficiency was 90% (9 / 10). Among the 6 fertilized eggs injected with sgRNA4 (No. 11-16), 4 developed to the morula stage, 2 even developed to the blastocyst stage, and 2 developed to the 8-16 cell stage. Cloning and sequencing found that gene mutations related to the sgRNA4 target sequence were detected in samples from 3 fertilized eggs, including base deletions and insertions, and the positive embryo knockout efficiency was 50% (3 / 6). The blank group (No. 17-18) developed to the 8-16 cell stage, and no mutations were detected.

[0033] Table 3 Embryo knockout efficiency of sgRNA2 and sgRNA4

[0034] The editing method in embryos is mainly knockout, such as Figure 1As shown in the figure, since sgRNA4 is far from the translation start site of the gene (targeting exon 16), it may lead to a certain amount of residual protein function (about 900aa), and the efficiency is relatively low (50%) in embryo verification, so sgRNA2, sgRNA6, and sgRNA7 and sgRNA8 targeting exon 3 are considered for further optimization.

[0035] 2.3.2 Targeting ATM sgRNA sequence optimization for genes It is reported that designing two sgRNAs for the same gene can improve the knockout efficiency. In the present invention, sgRNA2 and sgRNA6 were mixed with Cas9 protein as the first group (Cas9: sgRNA is 300ng / μL: 133ng / μL), and sgRNA7 and sgRNA8 were mixed with Cas9 protein as the second group (Cas9: sgRNA is 300ng / μL: 133ng / μL) and injected into 32 fertilized eggs in good condition. In addition, 3 blank fertilized eggs were selected as negative controls and cultured in vitro for one week.

[0036] Results: After injection of combination 1, 8 (15 in total) developed to the blastocyst stage or above, and the positive embryo knockout efficiency was 66.67% (10 / 15); if the undeveloped fertilized eggs (1 cell) were excluded, the positive embryo knockout efficiency was 90.9% (10 / 11). After injection of combination 2, 10 (17 in total) developed to the blastocyst stage or above, and the positive embryo knockout efficiency was 82.35% (14 / 17); if the undeveloped fertilized eggs (1 cell) were excluded, the positive embryo knockout efficiency was 100% (14 / 14). Combination 1 (sgRNA2 and sgRNA6) cuts DNA and mainly causes deletion or replacement of large fragments (>900bp) in the exon 6 and 7 regions of the ATM gene; combination 2 (sgRNA7 and sgRNA8) cuts DNA and mainly causes deletion of short sequences in exon 3 (~50bp) of the ATM gene. These mutations cause frameshift mutations. The gene target fragments and mutation series are shown in SEQ ID NO:9-15, respectively. In the fertilized eggs of the blank group, all developed to the 4-8 cell stage, and no mutations were detected. Combination 2 (sgRNA7 and sgRNA8) has a high knockout efficiency and is less toxic to embryos from the perspective of embryonic development. Combination 2 was selected for macaque embryos. ATM Knockout.

[0037] Example 2 Targeting Macaques ATM Gene knockout Healthy adult female macaques aged 6 to 15 years were selected as egg donors. After the eggs and sperm were collected, intracytoplasmic sperm injection was performed, and in vitro culture was performed to confirm the successful fertilization. After confirming the success of artificial insemination (the appearance of the second polar body), a mixture of Cas9 protein: sgRNA7 and sgRNA8 was injected. After in vitro culture, 30 to 60 well-developed transgenic embryos were selected. When the embryos developed to the 4-8 cell stage, artificial embryo transplantation was performed by microinjection. Healthy female macaques aged 6 to 15 years were selected as recipient monkeys, and high-quality cleavage embryos from the two-cell stage to the blastocyst stage were transplanted into the oviducts of 15 to 20 paired recipient female monkeys at a ratio of 2 to 3 embryos / recipient monkeys; 30 days after transplantation, macaques that did not observe menstruation underwent abdominal ultrasound confirmation, and confirmed pregnant female monkeys needed to be regularly examined, and two weeks before the expected date of delivery (gestational cycle is about 160 days-170 days). These operations rely on the transgenic technology platform of the Kunming Primate Center.

[0038] The recipient monkeys were confirmed to be pregnant with 3 babies by B-ultrasound, and no miscarriage or vaginal bleeding was observed during the pregnancy. Among the 3 recipient female monkeys, 2 gave birth to male baby monkey No. 1 and female baby monkey No. 3 through natural delivery, and 1 gave birth to male baby monkey No. 2 through caesarean section. 3 F0 generation babies to be identified were successfully obtained. ATM All knockout macaques survived, and their basic information is shown in Table 4. Three healthy newborn monkeys that were born close to the ATM knockout macaques (no more than one week before or after) were selected as control monkeys, of which monkey No. 2 and the control monkeys were artificially fed in an incubator.

[0039] Table 4 ATM Information table of knockout macaques and wild-type control macaques

[0040] Example 3 ATM Phenotypic characterization of knockout macaque models 3.1 ATM Genotyping of knockout macaques Through DNA sequencing analysis of the skin (derived from the ectoderm) and blood (derived from the mesoderm) of the three gene-edited macaques obtained above, the three gene-edited macaques (AT group) obtained were ATM Homozygous knockout of gene ATM - / - ). Figure 2 The genotypes and ATM protein expression of three transgenic macaques and their control macaques were shown. ATM Knockout macaques produced a 47-49 bp knockout ( Figure 2 ATM-mRNA was not detected in the AT group ( Figure 2 B) In addition, the expression of ATM protein could not be detected in the fibroblasts cultured from the ear margin tissue of the AT group ( Figure 2C), proving that the knockout was very complete, with no residual ATM protein. At the same time, the mutated sites were sequenced and analyzed. The sequencing results showed that all three gene-edited monkeys ATM Homozygous knockout ( ATM - / - ), the mutant sequences of the gene target fragments of AT macaques are shown in SEQ ID NO: 16-20. Among them, 49bp was knocked out in AT1 fibroblasts, and 18bp was inserted after knocking out 54bp in blood cells; 48bp fragments were knocked out in AT2 fibroblasts and blood cells; 47bp fragments were knocked out in AT3 fibroblasts and blood cells, which are consistent with the results of embryo verification. The severity of AT is related to ATM The mutation type of the gene is related to the residual amount of ATM protein. After optimizing the CRISPR system, the three AT macaques finally obtained were all ATM Homozygous knockout.

[0041] 3.2 ATM Identification of knockout macaque model phenotypes For the 3 ATM The phenotype of knockout macaques was analyzed. AT is a multi-system disease, but symptoms related to the nervous system are the most common clinical manifestations of AT, including Purkinje neuron and granule cell dysfunction, cerebellar atrophy, cerebellar ataxia, gait abnormalities, and fine motor impairment. At the same time, as the disease progresses, the patient's cognitive function may be impaired, which will affect executive function, language processing, working memory, spatial cognition, and emotional regulation, leading to overall intellectual disability. Other characteristics include capillary dilation of the conjunctiva, face, and brain; children are sensitive to ionizing radiation, immune cell dysfunction, metabolic system disorders, and susceptibility to cancer.

[0042] 3.2.1 ATM Eye examination of knockout macaques AT patients often have dilated eye capillaries and dilated blood vessels in the eyes and skin, which is an important sign for diagnosing AT patients. Compared with the WT group macaques, the AT group macaques showed symptoms of dilated blood vessels on the bulbar conjunctiva at 3-6 months of age, while no such symptoms were observed in the WT group ( Figure 3 This has not been observed in rodent models either.

[0043] 3.2.2 ATM Determination of growth and development of knockout macaques Characteristics of AT patients include short stature and developmental delay. The inventors evaluated the growth and development of macaques. Physical examinations were performed on macaques at 3, 6, 9, 12, and 15 months of age. The biparietal diameter of the macaques was determined by vernier calipers, and the head circumference, body length (the distance from the top of the head to the sole of the foot), leg length, arm length, chest circumference, abdominal circumference, thigh circumference, calf circumference, palm length, and foot length of the macaques were measured by a leather tape measure; the weight of the macaques was measured by an electronic scale, such as Figure 4-7 shown.

[0044] The results showed that at 3 months of age, there was no difference in weight between the two groups of macaques. As age increased, at 6, 9, and 12 months of age, the average weight of the AT group was significantly lower than that of the WT group; however, at 15 months of age, there was no significant difference in weight between the two groups of macaques (e.g. Figure 4 A). When AT3 macaques were observed individually, their body weight was significantly lower than that of WT macaques at every measurement point. This is similar to the characteristics of AT patients, including short stature and developmental delay. Figure 4 As shown in B, at 3 months of age, there was no difference in head circumference between the two groups of macaques. At 6, 9, 12, and 15 months of age, the average head circumference of the AT group was lower than that of the WT group and there was a significant difference. Figure 4 As shown in C, at 3 months of age, there was no difference in the biparietal diameters of the two groups of macaques; at 6, 9, 12, and 15 months of age, the average biparietal diameter of the AT group was lower than that of the WT group, and there was a significant difference. Figure 4 As shown in D, at 3 months of age, there was no difference in body length between the two groups of macaques. At 6, 9, 12, and 15 months of age, the average body length of the AT group was lower than that of the WT group, but there was no significant difference.

[0045] At the same time, the chest and abdominal circumferences of the two groups of macaques were evaluated. Figure 5 As shown in the figure, at 3 and 6 months of age, there was no difference in chest circumference between the AT and WT groups; at 9, 12, and 15 months of age, the chest circumference of the AT group was smaller than that of the WT group, but there was no significant difference. At 3 months of age, the abdominal circumference of the AT group was larger than that of the WT group, but there was no significant difference; at 6 months of age, there was no difference in abdominal circumference between the AT and WT groups; at 9, 12, and 15 months of age, the abdominal circumference of the AT group was smaller than that of the WT group, but there was no significant difference.

[0046] The upper limbs of the two groups of macaques were evaluated. Figure 6As shown. At 3-15 months of age, the arm length of the AT group was shorter than that of the WT group, but there was no significant difference between the two groups. At 6 and 9 months of age, the palm length of the AT group was lower than that of the WT group and there was a significant difference. At 15 months of age, the upper arm circumference of the AT group was lower than that of the WT group, with a significant difference; at 9 months of age, the lower arm circumference of the AT group was lower than that of the WT group, with a significant difference. In addition to the above points with significant differences, after 6 months of age, the palm length, upper arm circumference and forearm circumference of the AT group were all smaller than those of the WT group, but there was no significant difference.

[0047] The results of the evaluation of the lower limbs of the two groups of macaques are as follows: Figure 7 As shown in the figure, at 3 months of age, the leg length, thigh circumference and calf circumference of the AT group were longer than those of the WT group; after 6 months of age, the leg length, foot length, thigh circumference and calf circumference of the AT group were shorter than those of the WT group, but there was no significant difference between the two groups. At 9 months of age, the foot length of the AT group was lower than that of the WT group, and there was a significant difference.

[0048] In summary, the growth and development-related data of the AT group at 3 months of age were very similar to those of the WT group. However, with increasing age, growth retardation appeared in many data, and there were significant differences in some observation points.

[0049] 3.2.3 ATM Determination of blood-related indicators in knockout macaques Common symptoms in AT patients include primary immunodeficiency, frequent infections, elevated immune-related indicators, and abnormally elevated alpha-fetoprotein levels. 0.5-1 mL of blood was collected from the experimental macaques through the vein and placed in an EDTA-rinsed anticoagulant tube (purple tube). 20 μL of whole blood was drawn and analyzed by an automatic blood cell analyzer (Mindray BC-5000 Vet, veterinary use) to obtain data on cell components and proportions in the blood. The remaining blood was centrifuged at 3000 rpm at 4°C to remove the supernatant, and the immunoglobulin level and alpha-fetoprotein level related indicators were measured.

[0050] AT patients often have abnormal increase in white blood cells. Figure 8 A, At 3 months of age, there was no difference in the number of white blood cells between the AT and WT groups; at 6 months of age, it was seen that the white blood cell level of WT was normally increasing, and this phenomenon also occurred in human children: the level was high in early childhood, but there was no significant increase in the AT group; at 9 months of age (P=0.0341), 12 months of age (P=0.0182), and 15 months of age (P=0.0253), the white blood cell level of the WT group remained at a high level, but the AT group maintained an abnormally low value.

[0051] AT patients have a decrease in B lymphocytes and T cell lymphocytes. ATM The double-strand mutation of the gene is closely related. Figure 8B) In the WT group, lymphocytes showed a trend of first increasing (3-9 months) and then decreasing (9-15 months). In the AT group, lymphocytes remained at a low level at all ages, and were significantly different from the WT group at 6 months (P=0.0331) and 15 months (P=0.0004).

[0052] AT patients often develop leukemia, and a decrease in the number of red blood cells may be detected. At the five examination time points, there was no difference in the number of red blood cells between the AT group and the WT group (e.g. Figure 8 C), but AT3 is abnormally high in all checkpoints. Hemoglobin is an important reason why blood appears red, and it is also the target of red blood cells to bind oxygen or carbon dioxide. Figure 8 C) From 3 to 9 months of age, the hemoglobin levels of the AT and WT groups showed an increasing trend, but the hemoglobin level of the AT group was slightly lower than that of the WT group. This difference was statistically significant at 6 months of age (P=0.0246) and 12 months of age (P=0.0293). At the same time, the hemoglobin level of AT3 was significantly lower than that of all macaques, which was consistent with the law of hemoglobin changes. This is similar to the changes in leukemia that often occur in AT patients.

[0053] Immunoglobulins are mainly found in plasma and are important immune effector factors. They can be divided into five categories: IgG, IgA, IgM, IgD, and IgE. IgG is the most abundant component of immunoglobulins, accounting for about 70-75%. In humans, IgG begins to be synthesized in March after birth, gradually approaching adult levels, and plays a role in anti-infection. Comparison of the 3-month and 6-month data of the AT and WT groups ( Figure 8 D), IgG gradually increased and gradually stabilized after 6 months. However, the IgG level of AT was lower than that of the WT group at 9 months of age (P=0.0293); at 6 months of age (P=0.022), the IgG level of the AT group was inconsistent with that of the corresponding WT group. Although the mean values ​​were similar in the figure, there were significant differences within the groups. Although no IgG abnormalities were found in AT patients, it indicated that there were abnormalities in immune function. The abnormal IgG level of macaques in the AT group may be related to susceptibility to infection caused by immune abnormalities.

[0054] IgM is the earliest antibody synthesized and secreted during individual development and can be produced in the fetus in the late embryonic development. The detection of IgM in serum indicates a recent infection and can be used for early diagnosis of infection. Fig. 9As shown in A, at 9 months of age, the IgM level remained at the same level; however, at 3 months (P=0.119), 6 months (P=0.0118), 12 months (P=0.0324), and 15 months (P=0.0180), the IgM in the AT group was significantly higher than that in the WT group, and there was a significant difference. The increase in IgM may be similar to the results of IgA, which indicates a recent infection and may be related to the herpes-like infection occasionally seen in the AT group.

[0055] Human alpha-fetoprotein (AFP) is synthesized in the fetal liver, reaching its peak at 30 weeks of gestation, then gradually decreasing to approach adult levels at one year old. In AT patients, the level of alpha-fetoprotein is abnormally elevated. If alpha-fetoprotein is elevated in patients with recurrent infections and immunodeficiency, AT is highly suspected. Fig. 9 B shows that at 3 months of age, AFP levels in both the AT and WT groups remained high. From 3 to 9 months of age, the AFP level in the WT group decreased normally and remained at a low level from 9 to 15 months. However, the AFP level in the AT group decreased slightly from 3 to 6 months, but remained at a high level from 6 to 15 months, and showed significant differences from the WT group at 12 months (P=0.020) and 15 months (P=0.033).

[0056] 3.2.4 ATM Knockout macaque brain related analysis The neuropathological hallmarks of AT are diffuse degeneration or atrophy of the cerebellar vermis and hemispheres, hypoplasia of the inferior vermis, and reduced thickness of the hemispheric cortex, involving Purkinje neurons and a small number of granule cells. Neuropathological abnormalities are also observed in the cerebrum, brainstem, and spinal cord. The inventors tracked the brain volume and white matter changes of the two groups of monkeys through regular MRI.

[0057] The MRI results of 6-month-old and 12-month-old children were analyzed. Fig.10 As shown in the figure, the comparison of some sagittal sections of the cerebellum is shown. The left side is a cross section of the cerebellar hemisphere, where changes in the cerebellar cortex can be observed. The volume of the cerebellar cortex in the AT group is smaller than that in the control group. The right side is a comparison of the largest cross section, where changes in the volume and morphology of the cerebellar vermis can be observed. It was found that the cerebellar vermis in the AT group was significantly smaller than that in the WT group. At the same time, the volume was finely segmented, the volume of the cerebellar cortex was calculated, and the relative proportions of each time point to the standard atlas were preliminarily calculated by comparing with the standard atlas (see Table 5). The above results show that the cerebellar vermis and hemispheres of AT macaques are atrophied, while WT macaques do not have such symptoms.

[0058] Table 5 Cerebellar cortical volumes of macaques in the AT and WT groups and comparison with the standard cerebellum

[0059] 3.2.6 ATM Behavioral analysis of cerebellar motor function knockout in macaques In patients with cerebellar lesions, cerebellar lesions may lead to movement disorders, unstable standing, and ataxia symptoms. The present invention mainly performs 3D animal gait analysis.

[0060] The gait of the macaques was statistically analyzed and it was found that the gait cycle of the AT group was too short ( Fig.11 A), the corresponding is too fast a step frequency ( Fig.11 B). The stride length and stride radius of the AT group were shorter, which was significantly different from that of the WT group ( Fig.11 C and Fig.11 D). AT group in the support phase ( Fig.12 B, P<0.0001), swing phase ( Fig.12 C, P=0.0019) and the time of maintaining double-foot support ( Fig.12 D, P=0.0351) and WT group, the AT group had a very fast step frequency, and the time in the three states was very short, while the step interval ( Fig.12 A) There is no significant difference.

[0061] In summary, the method of the present invention produces 3 ATM - / - Compared with the control monkey (WT group), the AT group showed obvious AT symptoms of cerebellar atrophy, cerebellar dysfunction, conjunctival telangiectasia, growth retardation, immunosuppression and abnormally elevated alpha-fetoprotein levels. At the same time, the present invention also evaluated the learning, memory and fine activities of the two groups of macaques and found that at this stage (1-1.5 years old), the AT group did not show abnormalities in brain-related functions, which is consistent with the MRI results, that is, the degeneration at this stage only occurred in the cerebellum.

[0062] When analyzing the growth and development of macaques, the inventors found that the AT group had problems with growth retardation, and in addition to height and weight, there were significant differences in data such as head circumference; when analyzing the blood, the white blood cell level was abnormal, the lymphocyte ratio was abnormal, the hemoglobin level was abnormal, the alpha-fetoprotein level after birth was abnormally high, and the immunoglobulin IgG and IgM levels were abnormal. Another important phenotype is that the AT group had dilated veins on the conjunctiva of the eye at 3-6 months, which is very consistent with the phenotype of AT patients and is also an important indicator in the diagnosis of AT. In addition, the AT group had problems with motor function, including too wide a stride, unstable gait, and too fast a step frequency, which are consistent with human motor dysfunction. These results confirm that the AT macaques of the present invention simulate the phenotype of AT patients.

[0063] In summary, the present invention provides a method for targeting macaques ATM The sgRNA for gene knockout was successfully constructed ATM Gene knockout macaques, phenotypic analysis revealed: ATMThe knockout macaques showed typical symptoms of AT, such as cerebellar atrophy, movement disorders, conjunctival telangiectasia, growth retardation, immunosuppression, and abnormally elevated alpha-fetoprotein levels, which can effectively simulate the phenotype of human AT disease. In addition, core phenotypes such as cerebellar neurodegeneration did not appear in the rodent model, such as cerebellar vermis, hemispheric atrophy, and ataxia symptoms, or were difficult to observe, such as telangiectasia and fine motor movements. ATM Knockout macaques provide a unique resource for the analysis and mechanism of cerebellar-related neurodegeneration and behavioral phenotypes. ATM Gene-related diseases provide an ideal animal model, laying a foundation for understanding the pathogenic mechanisms of related diseases and for drug screening.

Claims

1. Targeting macaques ATM The sgRNA for gene knockout is characterized by: The nucleotide sequence of the sgRNA is at least one of the sequences shown in SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO:

8.

2. The targeted macaque as claimed in claim 1 ATM The sgRNA for gene knockout is characterized by: The nucleotide sequence of the sgRNA is a combination of SEQ ID NO: 2 and SEQ ID NO: 6 or a combination of SEQ ID NO: 7 and SEQ ID NO:

8.

3. The sgRNA according to claim 1 or 2 is used to target macaques ATM Application in gene knockout.

4. A macaque ATM The method for constructing a gene knockout model comprises the following steps: S1 will target macaques ATM Gene knockout sgRNA and Cas9 protein may target macaques ATM The knockout sgRNA and Cas9 mRNA mixture were co-injected into macaque embryos to obtain ATM Knockout macaque embryos; After in vitro screening, S2 was injected into the cells obtained from S1 by microinjection. ATM The knockout macaque embryos were transplanted into recipient female monkeys to produce ATM Knockout macaques.

5. The macaque according to claim 4 ATM Gene knockout model construction method ATM The application of gene knockout models in studying the occurrence and development mechanism of AT or screening therapeutic drugs for AT.

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