阅读说明：本技术 一种敲除诱导多能干细胞nans基因的方法和应用 (Method for knocking out NANS gene of induced pluripotent stem cell and application ) 是由 卜迁 岑小波 张华琴 代艳萍 于 2020-07-09 设计创作，主要内容包括：本发明公开了一种敲除诱导多能干细胞NANS基因的方法和应用,属于细胞模型构建领域。本发明采用电穿孔法将CRIPSR-Cas9基因编辑系统转入iPSC；将含有Puromycin抗性基因和GFP基因的Donor DNA插入NANS外显子,通过Puromycin对基因编辑后干细胞进行筛选；转染后加入CloneR<Sup>TM</Sup>培养细胞,该体系可以大大提高CRISPR-Cas9基因组编辑技术在干细胞中的基因编辑效率。本发明将有助于构建NANS缺陷型的3D大脑类器官模型,可应用于基于NANS突变引起的智力发育障碍发病机制的体外研究和药物研发。(The invention discloses a method for knocking out an induced pluripotent stem cell NANS gene and application thereof, belonging to the field of cell model construction. The CRIPSR-Cas9 gene editing system is transferred into iPSC by an electroporation method; inserting Donor DNA containing Puromycin resistance gene and GFP gene into NANS exon, and screening the gene edited stem cell by Puromycin; after transfection CloneR was added TM When the cell is cultured, the system can greatly improve the gene editing efficiency of the CRISPR-Cas9 genome editing technology in the stem cell. The invention will help to construct NANS-deficient 3D brain organoid models,can be applied to in vitro research and drug research and development of intellectual development disorder pathogenesis caused by NANS mutation.)

1. An sgRNA, comprising: the recognition sequence is TCTCGGCAATGATGAAGCAC.

2. A method for knocking out NANS gene in iPSC cell by CRISPR-CAS9 technology is characterized in that: the sgRNA recognition sequence is TCTCGGCAATGATGAAGCAC.

3. The method of claim 2, wherein: the method comprises the following steps:

1) transfecting the sgRNA-cas9 plasmid and the linear Donor DNA to iPSC, culturing, and screening successfully transfected positive iPSC;

2) selecting positive iPSC clone, and verifying through PCR, sequencing or protein detection;

the sgRNA-Cas9 plasmid of the step 1) carries sgRNA and Cas9 genes;

the sequence of the Donor DNA in the step 1) is shown as SEQ ID NO. 8.

4. The method of claim 3, wherein: the transfection in the step 1) is electrotransformation, and the conditions of electrotransformation are as follows:

voltage 1100V, pulse width 30ms, number of pulses 1.

5. The method of claim 4, wherein: electric conversion for useThe transfection system and the electrotransfer buffer solution are buffer.

6. The method of claim 4, wherein: step 1) after the end of electrotransformation, cells were added to a solution containing 10% CloneR^TMmTeSR of^TM1 culture in medium.

7. The method of claim 3, wherein: the sequence of the primer of the PCR in the step 2) is shown as SEQ ID NO. 9-12, wherein SEQ ID NO. 9-10 is the primer for identifying the wild type; 11-12 of SEQ ID NO are primers for identifying the mutant.

8. A cell produced by the method of any one of claims 2 to 7.

9. A method of making a NANS knockout brain organoid, comprising: induced differentiation of brain organoids using the cells of claim 8.

Technical Field

The invention relates to the field of cell model construction, in particular to a method for knocking out an inducible pluripotent stem cell NANS gene based on a CRISPR-CAS9 gene editing technology and application thereof.

Background

Intellectual Development Disorder (IDD), also known as Mental Retardation and Mental Retardation C Mental Retardation, is a group of diseases characterized by onset before 18 years of age, cognitive dysfunction (IQ <70 score), and deficits in social adaptation ability. It is also known as mental retardation (DD) before school age (under 5 years old) due to hypofunction of speech and motor development. The syndrome type intellectual impairment is often accompanied by congenital Multiple Malformations (MCA), special facial appearance, and the like. The etiology of ID/DD is complex, including environmental factors, perinatal hypoxia, and genetic factors, with the genetic factors accounting for 2/3. At present, because the pathogenesis of IDD is poorly understood, and the treatment of IDD is difficult, establishing IDD research and drug evaluation models is crucial to driving the progress of IDD treatment.

Induced Pluripotent Stem Cells (iPSCs) and genome editing technology are combined to establish a cell model, and a unique experimental platform is provided for disease research. By utilizing the platform system, researchers can research the influence of specific gene mutation and even chromosome structure variation on various cell types and tissue and organ functions of human beings and detailed molecular mechanisms thereof, and can establish personalized disease models carrying different genetic mutations for large-scale drug screening. The brain organoid is a cerebral cortex structure formed by the directional induction and differentiation of cells, can simulate the development process and the structural characteristics of the brain in early human embryo to a certain extent in vitro, can better keep the specific genotype and the protein expression level of a human body, and has great potential in the aspects of researching the origin and the pathology of mental diseases, screening drugs and modifying genes. Compared with the traditional animal research model, the appearance of human brain organoids reduces the species difference between the animal brain and the human brain, and provides a new tool for the in vitro research of mental diseases.

NANS, also known as sialic acid (phosphate) synthase (SAS), is responsible for encoding N-acetylneuraminic acid 9-phosphate synthase (NeuNAc-9-P synthase), which plays a key role in the primary synthesis of the most common sialic acid, N-acetylneuraminic acid (Neu5 Ac). Furthermore, as a key step in the pathway of production of the major sialylation substrate CMP-Neu5Ac, NANS can also affect sialylation of glycans, a modification that can mediate or modulate a variety of physiological and pathological processes, such as the correct establishment of the vertebrate embryonic nervous system, inflammatory and immune response pathways, tumorigenesis and metastasis in certain cancers. Recently, clinical studies have shown that biallelic mutations in NANS are associated with Intellectual Development Disorder (IDD), but the mechanism is not clear. The NANS-deficient cell line is established by the CRISPR-CAS9 technology, and is differentiated into brain organoids, so that an intellectual development disturbance in vitro disease model based on NANS mutation is expected to be established, the pathogenesis of intellectual development disturbance based on NANS mutation is further researched, and a medicine screening tool is provided for patients with intellectual development disturbance carrying NANS gene mutation.

At present, no report for constructing an intellectual development disturbance model based on NANS-deficient brain organoids exists.

Disclosure of Invention

The invention aims to solve the problems that: a method for knocking out an inducible pluripotent stem cell NANS gene based on a CRISPR-CAS9 gene editing technology and application thereof.

The technical scheme of the invention is as follows:

an sgRNA having the recognition sequence TCTCGGCAATGATGAAGCAC.

A method for knocking out NANS gene in iPSC cell by CRISPR-CAS9 technology is characterized in that: the sgRNA recognition sequence is TCTCGGCAATGATGAAGCAC.

The term "sgRNA recognition sequence" refers to: sequence of the genomic fragment recognized by the sgRNA.

The method as described above, comprising the steps of:

1) transfecting the sgRNA-cas9 plasmid and the linear Donor DNA to iPSC, culturing, and screening successfully transfected positive iPSC;

2) selecting positive iPSC clone, and verifying through PCR, sequencing or protein detection;

the sgRNA-Cas9 plasmid of the step 1) carries sgRNA and Cas9 genes;

the sequence of the Donor DNA in the step 1) is shown as SEQ ID NO. 2.

As the method, the transfection in the step 1) is electrotransfer, and the conditions of the electrotransfer are as follows:

voltage 1100V, pulse width 30ms, number of pulses 1.

The method as described above, usingThe transfection system and the electrotransfer buffer solution are E buffer.

As described above, step 1) after the electrotransformation has ended, the cells are added to a solution containing 10% CloneR^TMmTeSR of^TM1 culture in medium.

As the method, the primer sequence of the PCR in the step 2) is shown as SEQ ID NO 9-12, wherein SEQ ID NO 9-10 is a primer for identifying the wild type; 11-12 of SEQ ID NO are primers for identifying the mutant.

The cells prepared by the above method.

A method of making a NANS knockout brain organoid, comprising: it is induced and differentiated into brain organoids by using the cells.

Compared with the prior art, the invention has the following advantages:

(1) according to the invention, the NANS gene is knocked out from the iPSC cell by using the CRISPR/Cas9 system, the operation is simple and convenient, and the effect is complete and thorough. Through modifying and optimizing CRISPR/Cas9 genome editing technology, resistance gene Puromycin N-acetyl-transferase and GFP protein are co-expressed on a vector, and stem cells after gene editing are screened through PuromycinOptionally, the gene editing efficiency of the CRISPR/Cas9 genome editing technology in stem cells can be greatly improved, and the NANS knockout condition can be directly judged through GFP. Compared with a chemical or virus-mediated transfection mode, the invention adopts an electroporation mode to carry out transfection, thereby greatly improving the transfection efficiency. After transfection with CloneR^TMThe protection is carried out, and the survival capability and the clonogenic capability of the stem cells are improved. The optimized CRISPR/Cas9 genome editing technology is simple and convenient to operate, shortens time, improves efficiency and provides a powerful tool for future stem cell genome editing.

(2) Compared with methods such as silencing, knocking-down and interference, the NANS gene knocking-out method based on the CRISPR/Cas9 system has a more thorough knocking-out effect.

(3) According to the invention, the detection of gene and protein levels proves that NANS is successfully knocked out, which indicates that the protein is thoroughly changed and can cause complete loss of NANS function, and the cell line is verified to be differentiated into brain organoid, can be used for researching the influence of NANS mutation on brain development and is used for developing the medicine based on intellectual development disorder caused by NANS mutation.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 is a microscopic examination of GFP expressing positive cells after puromycin screening.

Fig. 2 is a diagram of the genotype identification of mutant cells transfected with CRISPR targeting vectors.

Fig. 3 is a graph of NANS knockout strategy and sequencing results of mutant cells.

Fig. 4 is a graph of the differences in expression levels of NANS protein between knock-out and wild-type cell lines detected by western blotting.

Fig. 5 shows the differentiation of NANS knockout cell lines into brain organoids.

Fig. 6 shows the structure of the NANS knockout brain organoid Vz region.

Detailed Description