SgRNA targeting rhesus monkey atm gene knockout and application

Abstract

The application discloses a sgRNA for targeting rhesus monkey ATM Gene knockout, and belongs to the technical field of biology. The sgRNA for targeting the rhesus monkey ATM gene is shown in any one of SEQ ID NO: 2, 6, 7 and 8. The application further provides a method for targeting and knocking out the rhesus monkey ATM gene by using the sgRNA: multiple sgRNAs are designed based on a CRISPR / Cas9 system and are targeted to the rhesus monkey ATM gene. ATM The sgRNA for knocking out the rhesus monkey ATM gene is mixed with a Cas9 mRNA mixture, and the mixture is jointly injected into a rhesus monkey embryo, and then the embryo is transplanted into a female rhesus monkey, so as to obtain ATM a rhesus monkey with the gene knocked out. The method provides a new method for establishing a non-human primate AT animal model, and provides a research basis for studying a cerebellum degenerative phenotype, pathological characteristics and inherent molecular mechanism of an AT patient, analyzing a pathogenic mechanism and drug screening.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to sgRNA for knocking out rhesus monkey ATM gene and application. BACKGROUND

[0002] Ataxia-telangiectasia (AT) is a rare autosomal recessive disease caused by the loss of ATM function, and its main features are cerebellar degeneration, extrapyramidal reaction, oculomotor nerve disorder, telangiectasia, immune dysfunction, endocrine disorder, 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 the molecular weight is 370 kD. ATM protein is a serine-threonine protein kinase related to DNA damage response and cell cycle regulation, which can phosphorylate more than 700 substrates, and is very important for accurately and effectively maintaining the genomic integrity of living organisms. Some AT patients have severe ataxia and cerebellar atrophy in early stage, and coordination of eyeball is impaired after ten years old, reading becomes more and more difficult, accompanied by fine motor dysfunction, such as writing, coloring and eating with utensils, and symptoms such as easy fatigue and difficulty concentrating.

[0003] Although the prior art ATM Although the prior art

[0004] In recent years, the CRISPR / Cas9 system has rapidly become a leading gene editing tool with the advantages of high efficiency, simple operation and low cost, and has been widely used in the fields of gene function research, disease model and gene therapy. After searching, there is no report on knocking out rhesus monkey ATM gene based on the CRISPR / Cas9 system, and there is no report on sgRNA for knocking out rhesus monkey ATM gene. SUMMARY

[0005] To solve the above technical problems, the present application provides sgRNA for targeting rhesus monkey ATM knockout and application. The technical solutions of the present application are as follows:

[0006] The present application provides sgRNA for targeting rhesus monkey ATM 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.

[0007] 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.

[0008] The present application also protects the application of the sgRNA in targeting rhesus monkey ATM knockout.

[0009] The present application also protects a method for constructing a rhesus monkey ATM knockout model, comprising the following steps:

[0010] S1: co-injecting sgRNA for targeting rhesus monkey ATM knockout and Cas9 protein or co-injecting sgRNA for targeting rhesus monkey ATM knockout and Cas9 mRNA mixture into rhesus monkey embryos to obtain rhesus monkey embryos with ATM knockout;

[0011] S2: after in vitro screening, transplanting the rhesus monkey embryos with ATM knockout obtained in S1 into a recipient female monkey to produce a rhesus monkey with ATM knockout.

[0012] The present application also protects the application of the rhesus monkey ATM knockout model in studying the mechanism of occurrence and development of AT or screening therapeutic drugs for AT.

[0013] Compared with the prior art, the present application has the following beneficial effects:

[0014] The present application designs and screens a CRISPR / Cas9 knockout system for rhesus monkey ATM genes, provides sgRNA for targeting rhesus monkey ATM knockout, and successfully constructs a rhesus monkey with ATM knockout. Phenotype analysis shows that: ATMThe gene knockout macaques have obvious cerebellar atrophy, motor disorders, conjunctival capillary dilation, growth retardation, immunodeficiency and abnormal elevation of alpha-fetal protein level, which are consistent with the typical symptoms of AT patients, and better simulate the disease phenotypes of early AT in humans. Among them, some core phenotypes do not appear in rodent models, such as cerebellar vermis atrophy, hemisphere atrophy and ataxia symptoms, or phenotypes that are not convenient to observe, such as capillary dilation and fine motor skills. The method of the present application provides a method for constructing an animal model of diseases related to genes such as AT related diseases, and provides a research basis for understanding the pathogenesis and drug screening. ATM The method of the present application provides a method for constructing an animal model of diseases related to genes such as AT related diseases, and provides a research basis for understanding the pathogenesis and drug screening. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The cutting effects of two groups of sgRNA are shown in the following table, wherein Figure 1 A is the cutting effect of combination 1 (sgRNA2+sgRNA6), Figure 1 B is the cutting effect of combination 2 (sgRNA7+sgRNA8).

[0016] Figure 2 The cutting effects of two groups of sgRNA are shown in the following table, wherein ATM The genotype and ATM protein expression of the knockout macaques are shown in the following table, wherein Figure 2 A shows the target gene mutation of the AT group; Figure 2 B is ATM The ATM-mRNA detection situation of the knockout macaques is shown in the following table; Figure 2 C shows the ATM protein expression in the fibroblasts of the AT group.

[0017] Figure 3 The contrast diagram of conjunctival capillary dilation of macaques is shown in the following table.

[0018] Figure 4 The body development comparison diagram of the WT group and the AT group is shown in the following table, wherein Figure 4 A is the body weight comparison diagram of the WT group and the AT group, *: P<0.05; Figure 4 B is the head circumference comparison diagram of the WT group and the AT group, *: P<0.05, Figure 4 C is the bispinales comparison diagram of the WT group and the AT group, *: P<0.05; Figure 4 D is the body length comparison diagram of the WT group and the AT group, **: P<0.01, paired t-test (two-tailed), mean±SEM.

[0019] Figure 5 The chest circumference and abdominal circumference comparison diagram of the WT group and the AT group is shown in the following table, wherein Figure 5 A is the abdominal circumference comparison diagram of the WT group and the AT group, Figure 5 B is the abdominal circumference comparison diagram of the WT group and the AT group. Paired t-test (two-tailed), mean±SEM.

[0020] Figure 6 Figure 8 is a graph comparing upper limbs of WT and AT groups, where Figure 6 A-D are graphs comparing arm length, palm length, upper arm circumference, and lower arm circumference of AT and WT groups, respectively. Paired t-test (two-tailed), 9 months of age, Ratio paired t-test correction, mean ± SEM, ***: P < 0.001, *: P < 0.05.

[0021] Figure 7 Figure 9 is a graph comparing lower limbs of WT and AT groups, where Figure 7 A-D are graphs comparing leg length, foot length, upper leg circumference, and lower leg circumference of WT and AT groups, respectively, **: P < 0.01.

[0022] Figure 8 Figure 10 is a graph comparing blood analysis of WT and AT groups of rhesus monkeys, where Figure 8 A is a graph comparing white blood cell levels of WT and AT groups, *: P < 0.05; Figure 8 B is a graph comparing lymphocyte levels of WT and AT groups, ***: P < 0.001, *: P < 0.05; Figure 8 C is a graph comparing hemoglobin levels of WT and AT groups, *: P < 0.05; Figure 8 D is a graph comparing immunoglobulin IgG levels of WT and AT groups, *: P < 0.05, where 6 months of age is corrected by Ratio paired t-test, paired t-test (two-tailed), mean ± SEM.

[0023] Figure 9 Figure 11 is a graph comparing immune levels of WT and AT groups of rhesus monkeys, Figure 9 A is a graph comparing immunoglobulin IgM levels of WT and AT groups, paired t-test (two-tailed), mean ± SEM, *: P < 0.05; Figure 9 B is a graph comparing alpha-fetoprotein levels of WT and AT groups, paired t-test (two-tailed), mean ± SEM, *: P < 0.05, as the WT group values were too small at 12 and 15 months of age, the difference between the two groups was not equal, and the Ratio paired t-test correction was used.

[0024] Figure 10 Figure 12 is a graph comparing sagittal sections of the brain of AT and WT monkeys of 6 and 12 months of age, where the left graph shows the comparison of the cerebellar hemispheres of rhesus monkeys, and the right graph shows the comparison of the maximum section, showing the comparison of the cerebellar vermis.

[0025] Figure 11 Figure 13 is a graph comparing gait cycle, stride, step frequency, and step length, where Figure 11 A is a graph comparing gait cycle statistics of WT and AT groups, Figure 11 B is a graph comparing step frequency statistics, Figure 11 C is a graph comparing stride, Figure 11D is the step length comparison chart, Figure 11 A-C: Mann-Whitney rank sum test (two-tailed); Figure 11 D: Welch's test correction (two-tailed), all expressed as mean ± SEM, **: P < 0.01, *: P < 0.05.

[0026] Figure 12 WT group and AT group of rhesus monkey step interval and dynamic gait comparison chart, wherein Figure 12 A is a statistical comparison chart of step interval of WT group and AT group of rhesus monkeys, Figure 12 B is a comparison chart of support phase, Figure 12 C is a comparison chart of swing phase, Figure 12 D is a comparison chart of double foot standing. Figure 12 A: Welch's test correction (two-tailed), Figure 12 B-D: unpaired t-test (two-tailed), mean ± SEM, ****: P < 0.0001, **: P < 0.01, *: P < 0.05. DETAILED DESCRIPTION

[0027] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with the drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0028] Experimental animals and experimental reagents: The rhesus monkey donors and recipients used in this experiment are from the Kunming Primate Research Center (KPRC) of the Chinese Academy of Sciences, and are fed according to standard procedures. All embryos in the experiment are from KPRC. The Cos7 cell line (African green monkey kidney cells) sample used in the experiment is from the Kunming Cell Bank of the Chinese Academy of Sciences. Vector selection Cos7 cell line plasmid system: pSpCas9(BB)-2A-GFP.

[0029] Example 1 Targeting rhesus monkeys based on CRISPR / Cas9 system ATM Design and screening of sgRNA for gene knockout

[0030] Analysis of rhesus monkey ATM gene sequence by bioinformatics method, and design specific sgRNAs combined with mature CRISPR target point evaluation software. First, preliminary screening is carried out in vitro using Cos7 cell line, the screened sgRNA is further verified in fertilized eggs, and after determining the knockout efficiency, transplantation is carried out.

[0031] 1. sgRNA design

[0032] For rhesus monkeys ATMSeveral major exons were designed, and multiple specific sgRNAs were designed. 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, preferably with an sgRNA that differs 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 eight designed target sequences are shown in Table 1 below, and their nucleotide sequences are shown in SEQ ID NOs: 1-8:

[0033] Table 1 sgRNA sequence list

[0034]

[0035] 2. Knockout based on CRISPR / Cas9 system ATM Screening of sgRNA for genes

[0036] 2.1 In vitro transcription and synthesis of Cas9 mRNA and sgRNA

[0037] Cas9 mRNA and sgRNA were in vitro transcribed using a T7 in vitro transcription kit. To prepare Cas9 mRNA and sgRNAs, the T7 promoter sequence was added to the Cas9 or sgRNA template via PCR amplification. The T7-Cas9 / sgRNA PCR product was gel-purified and in vitro transcribed using a commercial T7 in vitro transcription kit (Thermo, K0441). Cas9 mRNA and sgRNA were purified using a MinElute PCR purification kit. After quality control, the final concentration of the Cas9 mRNA was 200 ng / μL and stored at -80°C. The final concentration of the sgRNA was 20 ng / μL and stored at -80°C.

[0038] 2.2 Detection of knockout efficiency of sgRNA 1-6 in Cos7 cells

[0039] A plasmid vector (pSpCas9(BB)-2A-GFP) encoding sgRNAs 1-6 was constructed and transiently transfected into Cos7 cells. The editing efficiency was assessed by TA cloning. The results are shown in Table 2.

[0040] Table 2 sgRNA 1-6 knockout efficiency comparison table in Cos7 cells

[0041]

[0042] From Table 2, the knockout efficiency of sgRNA 1-6 in Cos7 cells can be seen, the number of detections is randomly selected clones for sequencing, the transfection efficiency is roughly estimated by comparing the positive cells after transfection, and the mutation rate is (number of mutations / detection number) / transfection efficiency. It can be seen that the in vitro efficiency of sgRNA2, sgRNA4 and sgRNA6 is higher, all of which are more than 50%.

[0043] 2.3 sgRNAs knockout efficiency in embryos

[0044] 2.3.1 sgRNA2 and sgRNA4 knockout efficiency in embryos

[0045] The sgRNA2, sgRNA4 and Cas9 mRNA mixed solution obtained above were respectively injected into rhesus monkey embryos by embryo microsurgery technique, and the development of the embryos after injection was observed, and the knockout efficiency was detected. At the time of injection, the Cas9 mRNA was mixed with the two kinds of sgRNA, and the concentration was controlled by using nuclease-free water. Specifically: Cas9 mRNA / sgRNA2 (1-10) or sgRNA4 (11-16) (wherein the concentration of Cas9 mRNA is 100 ng / μL, and the concentration of sgRNA2 and sgRNA4 is 10 ng / μL). Injected into 16 fertilized eggs in good condition, of which 11 were in MII phase, 0 were in MI phase, and 5 were in GV phase, and cultured in vitro for one week. In addition, 2 blank fertilized eggs were selected as negative controls.

[0046] The results are shown in Table 3. Among the 10 fertilized eggs injected with sgRNA2 (1-10), 6 developed to the morula stage, even 4 developed to the blastocyst stage, and 4 developed to the 8-16 cell stage. Cloning sequencing found that gene mutations related to sgRNA2 target sequence were detected in samples from 9 fertilized eggs, including base deletion and insertion, and the positive embryo knockout efficiency was 90% (9 / 10). Among the 6 fertilized eggs injected with sgRNA4 (11-16), 4 developed to the morula stage, even 2 developed to the blastocyst stage, and 2 developed to the 8-16 cell stage. Cloning sequencing found that gene mutations related to sgRNA4 target sequence were detected in samples from 3 fertilized eggs, including base deletion and insertion, and the positive embryo knockout efficiency was 50% (3 / 6). The blank group (17-18) developed to the 8-16 cell stage, and no mutation was detected.

[0047] Table 3 sgRNA2 and sgRNA4 embryo knockout efficiency

[0048]

[0049] The editing method in the embryo is mainly knockout, as shown in the following table. Since sgRNA4 is far from the translation start site of the gene (for exon 16), it can cause some residual protein function (about 900 aa), and the efficiency is relatively low (50%) in the embryo verification, so sgRNA2, sgRNA6, and sgRNA7 and sgRNA8 for exon 3 are considered for further optimization. Figure 1

[0050] 2.3.2 Targeting ATM Optimization of sgRNA sequence of the gene

[0051] It is reported that designing two sgRNAs for the same gene can improve the knockout efficiency. The present application mixes sgRNA2 and sgRNA6 with Cas9 protein as the first group (Cas9: sgRNA is 300 ng / μL: 133 ng / μL), and mixes sgRNA7 and sgRNA8 with Cas9 protein as the second group (Cas9: sgRNA is 300 ng / μL: 133 ng / μL) to inject into 32 well fertilized eggs, and select 3 blank fertilized eggs as negative controls, and culture in vitro for one week.

[0052] Results: After injecting combination 1, 8 (total of 15) developed to the blastocyst stage and above, the positive embryo knockout efficiency was 66.67% (10 / 15); if excluding the non-developing fertilized eggs (1 cell), the positive embryo knockout efficiency was 90.9% (10 / 11). Injection of combination 2, 10 (total of 17) developed to the blastocyst stage and above, the positive embryo knockout efficiency was 82.35% (14 / 17); if excluding the non-developing fertilized eggs (1 cell), the positive embryo knockout efficiency was 100% (14 / 14). Combination 1 (sgRNA2 and sgRNA6) mainly forms deletion or replacement of a large fragment (>900 bp) in the 6th and 7th exon regions of the ATM gene after cutting DNA; combination 2 (sgRNA7 and sgRNA8) mainly causes deletion of a short sequence (~50 bp) in the 3rd exon of the ATM gene after cutting DNA, which causes a frameshift mutation. The gene target fragment and mutation series are shown in SEQ ID NO: 9-15. In the blank group of fertilized eggs, all developed to the 4-8 cell stage, and no mutation was detected. Combination 2 (sgRNA7 and sgRNA8) has higher knockout efficiency and less toxicity to the embryo from the perspective of embryo development, so combination 2 is selected for rhesus monkey embryo knockout. ATM

[0053] Example 2 Targeting rhesus monkey ATM ​​Gene knockout

[0054] Healthy adult female macaques aged 6 to 15 years were selected as egg donors. After egg and sperm retrieval, intracytoplasmic sperm injection (ICI) was performed. After in vitro culture, successful fertilization was confirmed by the appearance of a zygote. After confirmation of successful artificial insemination (appearance of a 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 reached the 4-8 cell stage, artificial embryo transfer (AIET) was performed via microinjection. Healthy female macaques aged 6 to 15 years were selected as recipients. High-quality cleavage embryos from the two-cell to blastocyst stage were transferred into the oviducts of 15 to 20 paired recipient females at a ratio of 2 to 3 embryos per recipient monkey. Thirty days after transfer, macaques not yet menstruating underwent abdominal ultrasound confirmation. Pregnant females were regularly examined, beginning two weeks before the expected date of birth (gestational age approximately 160-170 days). These procedures were performed using the transgenic technology platform at the Kunming Primate Center.

[0055] The recipient monkey was confirmed to be pregnant with 3 babies by B-ultrasound. 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 offspring were successfully obtained and are awaiting identification. ATM All knockout macaques survived, and their basic information is shown in Table 4. Three healthy newborn macaques born close to the ATM knockout macaques (no more than one week before or after) were selected as controls. Both monkey No. 2 and the control monkeys were artificially raised in an incubator.

[0056] Table 4 ATM Information table of knockout macaques and wild-type control macaques

[0057]

[0058] Example 3 ATM Phenotypic characterization of knockout macaque models

[0059] 3.1 ATM Knockout macaque genotype identification

[0060] By sequencing and analyzing the DNA 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 gene knockout ( ATM - / - ). Figure 2 The genotypes and ATM protein expression of three transgenic macaques and their control macaques were shown. ATM Knockout in macaques resulted in a 47-49 bp knockout ( Figure 2A), no ATM-mRNA in AT group could be detected ( Figure 2 B), and no ATM protein expression in fibroblasts from ear margin tissue of AT group could be detected either ( Figure 2 C), proving that the knockout is very complete, with no residual ATM protein. At the same time, the site of the mutation was sequenced. The sequencing results show that the three gene-edited macaques are all ATM homozygous knockouts ( ATM - / - ), and the mutation sequences of the gene target fragments of the AT macaques are shown in SEQ ID NO: 16-20, respectively. Among them, 49 bp of the fibroblasts of AT1 were knocked out, and 18 bp of the blood cells were inserted after 54 bp of the knockout; 48 bp of the fibroblasts and blood cells of AT2 were knocked out; 47 bp of the fibroblasts and blood cells of AT3 were knocked out, which is consistent with the results in the embryo verification. The severity of AT is related to the type of gene mutation and the amount of residual ATM protein, and after optimization of the CRISPR system, the three AT macaques obtained are all ATM homozygous knockouts. ATM

[0061] 3.2 ATM Phenotype identification of knockout macaque models

[0062] The phenotypes of the three ATM knockout macaques obtained were analyzed. AT is a multisystem disease, but the 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 the appearance of fine motor impairment, and as the disease progresses, the patient's cognitive function may be impaired, affecting executive function, language processing, working memory, spatial cognition, and emotional regulation, leading to overall intellectual impairment, other features include telangiectasia of the conjunctiva, face, and brain capillaries; children are sensitive to ionizing radiation, have defective immune cell function, have metabolic system disorders, and are prone to cancer, etc.

[0063] 3.2.1 ATM Eye examination of knockout macaques

[0064] AT patients often have eye telangiectasia, and eye and skin blood vessels are dilated, which is an important marker for diagnosing AT patients. After detection, compared with the WT group of macaques, the blood vessels in the conjunctiva of the AT group of macaques were dilated 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.

[0065] 3.2.2 ATM Determination of the growth and development of knockout macaques​

[0066] AT patient characteristics include short stature, developmental delay, etc. The inventors assessed the growth and development of the macaques. Physical examinations were performed at 3 months, 6 months, 9 months, 12 months, and 15 months of age. Bitemporal diameter was determined by vernier caliper. Head circumference, standing height (distance from the top of the head to the floor), leg length, arm length, chest circumference, abdominal circumference, thigh circumference, calf circumference, hand length, and foot length were measured by tape measure. Body weight was measured by electronic scale, as shown in Figure 4-7

[0067] It was found that at 3 months of age, there was no difference in body weight between the two groups of macaques. As the age increased, the average body weight of the AT group was lower than that of the WT group at 6 months, 9 months, and 12 months, and the difference was significant; but at 15 months of age, there was no significant difference in body weight between the two groups of macaques (as shown in Figure 4 A). When observing AT3 macaques alone, it was found that at each measurement point, the body weight was significantly lower than that of WT monkeys. This is similar to the characteristics of AT patients including short stature, developmental delay. As shown in Figure 4 B, at 3 months of age, there was no difference in head circumference between the two groups of macaques, and at 6 months, 9 months, 12 months, and 15 months, the average head circumference of the AT group was lower than that of the WT group and the difference was significant. As shown in Figure 4 C, at 3 months of age, there was no difference in bitemporal diameter between the two groups of macaques; at 6 months, 9 months, 12 months, and 15 months, the average bitemporal diameter of the AT group was lower than that of the WT group, and the difference was significant. As shown in Figure 4 D, at 3 months of age, there was no difference in standing height between the two groups of macaques, and at 6 months, 9 months, 12 months, and 15 months, the average standing height of the AT group was lower than that of the WT group, but there was no significant difference.

[0068] At the same time, the chest circumference and abdominal circumference of the two groups of macaques were assessed. As shown in Figure 5 , at 3 months and 6 months of age, there was no difference in chest circumference between the AT group and the WT group of monkeys; at 9 months, 12 months, 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 group and the WT group of monkeys; at 9 months, 12 months, 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.

[0069] The upper limbs of the two groups of macaques were assessed, and the results are shown in Figure 6 ​Figure 6 shows the results of the evaluation of the lower limbs of the two groups of macaques. 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 shorter than that of the WT group, and there was a significant difference.

[0070] Figure 6 shows the results of the evaluation of the lower limbs of the two groups of macaques. 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 shorter than that of the WT group, and there was a significant difference. Figure 7

[0071] In summary, the growth and development-related data of the AT group at 3 months of age were similar to those of the WT group, but as the age increased, the growth and development of the AT group showed signs of retardation in many data points, and there were significant differences in some observation points.

[0072] 3.2.3 ATM Measurement of blood-related indicators in knockout macaques

[0073] In AT patients, primary immunodeficiency, frequent infections, elevated immune-related indicators, and abnormal elevation of alpha-fetoprotein levels are common. 0.5-1 mL of blood was collected from the experimental macaques by venous sampling and placed in an EDTA-coated anticoagulant tube (purple tube). 20 μL of whole blood was taken and analyzed by a fully automatic blood cell analyzer (Mindray BC-5000 Vet, veterinary use) to obtain the cell composition and proportion data 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.

[0074] AT patients often have abnormal elevation of white blood cells. As shown in Figure 7A, 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, the white blood cell level of the WT group was normally elevated, which is also observed in human children: high levels in early childhood, but the AT group did not show significant elevation. At 9 months of age (P=0.0341), 12 months of age (P=0.0182), and 15 months of age (P=0.0253), the WT group maintained a high level of white blood cells, but the AT group maintained an abnormally low value. Figure 8 AT patients have reduced B lymphocytes, and T lymphocyte cells are also often reduced, and the reduction of lymphocytes in AT is often associated with

[0075] double-stranded mutations in the gene. In all observation points (e.g., Figure 7B), the number of B lymphocytes in the AT group was significantly lower than that in the WT group, and the difference was statistically significant. ATM Figure 8 ​​B), 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 of all ages maintained a low level, and there were significant differences with the WT group at 6 months (P=0.0331) and 15 months (P=0.0004).

[0076] AT patients often have leukemia, and a decrease in the number of red blood cells can be detected. There was no difference in the number of red blood cells between the AT and WT groups at the 5 examination time points (e.g. Figure 8 C), but AT3 was abnormally high at all examination points. Hemoglobin is an important reason for the redness of blood, and is also the target site for red blood cells to bind oxygen or carbon dioxide. At the examination points (e.g. Figure 8 C), the hemoglobin levels of the AT and WT groups showed an increasing trend from 3-9 months of age, but the hemoglobin levels of the AT group were slightly lower than those of the WT group, and the difference was statistically significant at 6 months (P=0.0246) and 12 months (P=0.0293). At the same time, the hemoglobin levels of AT3 were significantly lower than those of all macaques, consistent with the pattern of hemoglobin changes. This is similar to the changes that often occur in AT patients with leukemia.

[0077] Immunoglobulins are mainly present in plasma and are important immune effectors, which can be divided into IgG, IgA, IgM, IgD and IgE. IgG is the most common component of immunoglobulins, accounting for about 70-75%. In humans, IgG begins to be synthesized at 3 months of age and gradually approaches adult levels, playing a role in anti-infection. By comparing the 3-month and 6-month data of the AT and WT groups (e.g. Figure 8 D), it was found that IgG gradually increased and stabilized after 6 months. However, the IgG levels of the AT group were lower than those of the WT group at 9 months (P=0.0293); at 6 months (P=0.022), the IgG levels of the AT group were inconsistent with those of the corresponding WT group, although the means were similar, but there were significant differences within the groups. Although no IgG abnormalities were found in AT patients, it indicates that there are abnormalities in immune function, and the abnormal IgG levels of macaques in the AT group may be related to the susceptibility to infection caused by immune abnormalities.

[0078] IgM is the earliest antibody to be synthesized and secreted during individual development, and can be produced in fetuses in the late embryonic development. The detection of IgM in serum suggests a recent infection and can be used for early diagnosis of infection. For example, Figure 9As shown in Figure A, IgM levels remained essentially constant at 9 months of age; however, at 3 months (P=0.119), 6 months (P=0.0118), 12 months (P=0.0324), and 15 months (P=0.0180), IgM levels in the AT group were significantly higher than those in the WT group, with significant differences. Similar to the IgA results, the increase in IgM suggests recent infection, possibly related to the occasional herpes-like infection seen in the AT group.

[0079] 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 by one year of age. In patients with AT, the level of AFP is abnormally elevated. If AFP is elevated in patients with recurrent infections and immunodeficiency, AT is highly suspected. Figure 9 As shown in Figure B, at 3 months of age, AFP levels in both the AT and WT groups remained high. The AFP level in the WT group decreased normally from 3 to 9 months of age 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).

[0080] 3.2.4 ATM Knockout macaque brain correlation analysis

[0081] The neuropathological hallmarks of AT are diffuse degeneration or atrophy of the cerebellar vermis and hemispheres, hypoplasia of the inferior vermis, and reduced hemispheric cortical thickness, involving Purkinje neurons and a small number of granule cells. Neuropathological abnormalities have also been observed in the cerebrum, brainstem, and spinal cord. The inventors tracked changes in brain volume and white matter in both groups of monkeys using regular MRI.

[0082] The MRI results of 6-month-old and 12-month-old children were analyzed. Figure 10 The figure shows a comparison of partial sagittal sections of the cerebellum. The left side shows a cross-section of the cerebellar hemisphere, revealing changes in the cerebellar cortex. The cerebellar cortex volume in the AT group is smaller than that in the control group. The right side shows a comparison of the largest cross-section, revealing changes in the volume and morphology of the cerebellar vermis. The cerebellar vermis in the AT group is significantly smaller than that in the WT group. Furthermore, fine-scale segmentation was performed to calculate the cerebellar cortical volume. Comparisons were made to a standard atlas, and preliminary calculations of the relative proportions of each time point to the standard atlas were performed (see Table 5). These results indicate that AT monkeys exhibit atrophy of the cerebellar vermis and hemispheres, while WT monkeys do not exhibit such atrophy.

[0083] Table 5 Cerebellar cortical volumes of macaques in the AT and WT groups and comparison with the standard cerebellum

[0084]

[0085] 3.2.6 ATMKnockout of cerebellar motor function behavior analysis of macaques

[0086] In patients with cerebellar lesions, lesions of the cerebellum can cause motor disorders, and symptoms of unsteady standing and ataxia. The present application mainly carries out 3D animal gait analysis.

[0087] The gait of macaques was statistically analyzed, and it was found that the AT group showed a short gait cycle ( Figure 11 A), and correspondingly, the step frequency was too fast ( Figure 11 B). The step length and step radius of the AT group were both shorter than those of the WT group, and there were significant differences ( Figure 11 C and Figure 11 D). The AT group had significant differences in support phase ( Figure 12 B, P<0.0001), swing phase ( Figure 12 C, P=0.0019) and double foot support time ( Figure 12 D, P=0.0351) compared with the WT group. The AT group had a very short time in the three states because of the fast step frequency, and there was no significant difference in step interval ( Figure 12 A).

[0088] In summary, the method of the present application produces 3 ATM - / - Compared with the control monkeys (WT group), the AT group showed obvious symptoms of cerebellar atrophy, cerebellar dysfunction, conjunctival capillary dilation, growth retardation, immunosuppression and abnormal elevation of alpha-fetal protein level. At the same time, the learning and memory and fine activities of the two groups of macaques were evaluated, and it was found that the AT group did not show abnormal brain-related functions at this stage (1-1.5 years old), which is consistent with the MRI results, i.e. the degeneration only occurs in the cerebellum at this stage.

[0089] When analyzing the growth and development of macaques, the inventors found that the AT group had growth and development retardation, and there were significant differences in head circumference and other data besides height and weight; when analyzing the blood, the white blood cell level was abnormal, the lymphocyte proportion was abnormal, the hemoglobin level was abnormal, the alpha-fetal protein level was abnormally high after birth, and the immunoglobulin IgG and IgM levels were abnormal. Another important phenotype is that the AT group has venous dilation on the conjunctival of the eyeball at 3-6 months, which is very consistent with the phenotype of AT patients and is an important indicator in the diagnosis of AT. In addition, the AT group has problems in motor function, including wide step width, unstable gait, and fast step frequency, which are consistent with human motor dysfunction. These results confirm that the AT macaques of the present application simulate the phenotype of AT patients.

[0090] In summary, the present application provides sgRNA for targeted knockout of macaques ATM , which is successfully constructedATM Gene knockout macaques, phenotype analysis found: ATM Gene knockout macaques appear cerebellar atrophy, dyskinesia, conjunctival telangiectasia, growth retardation, immunodeficiency and abnormal elevation of alpha-fetoprotein level and other typical symptoms of AT, which can effectively simulate the human AT disease phenotype. Moreover, the core phenotype such as cerebellar neurodegeneration has not appeared in rodent models, such as cerebellar vermis, hemispheric atrophy and ataxia symptoms, or is not convenient to observe, such as telangiectasia and fine motor skills. The gene knockout macaques constructed by the present application provide a unique resource for the analysis and mechanism analysis of cerebellum-related neurodegeneration and behavior phenotype, and provide an ideal animal model for studying ATM Gene knockout macaques, which provide a unique resource for the analysis and mechanism analysis of cerebellum-related neurodegeneration and behavior phenotype, and provide an ideal animal model for studying ATM Gene-related diseases, and provide a basis for understanding the pathogenesis of related diseases and drug screening.

Claims

1. A method for constructing a macaque ataxia-telangiectasia syndrome (AT) model, comprising the following steps: S1 will target macaques ATM Gene knockout sgRNA and Cas9 protein may target macaques ATM The mixture of gene knockout sgRNA and Cas9 mRNA was co-injected into macaque fertilized eggs to obtain ATM Gene knockout macaque embryo; the Cas9:sgRNA ratio is 300 ng / μL:133 ng / μL; After in vitro screening, S2 was injected into the S1-derived ATM The gene knockout macaque embryos were transplanted into recipient female monkeys to produce ATM Knockout macaques; The nucleotide sequence of the sgRNA is a combination of SEQ ID NO: 7 and SEQ ID NO:

8.

2. Use of the AT model constructed by the method for constructing a macaque ataxia-telangiectasia syndrome (AT) model according to claim 1 in studying the occurrence and development mechanism of AT or screening therapeutic drugs for AT.

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