CN116042710A - Method for specifically modifying monkey-derived NOVA1 gene into human gene - Google Patents

Abstract

The invention belongs to the field of genetic engineering and genetic modification, and provides a method for modifying a monkey-source NOVA1 gene into a humanized gene, which comprises the following steps: determining a specific site for modifying a monkey cell NOVA1 genome into a human gene and the sgRNA, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1; the combined sequence of U6-sgRNA-scafaold containing the sgRNA is synthesized, the puromycin-containing fragment is amplified, and the puromycin-containing fragment is constructed on an original vector pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP to form a final vector pCMV-T7-ABEmax (7.10) -NOVA1. The invention realizes the site-directed mutation of the NOVA1 gene and provides a possible technical means and clinical thought for researching specific sites of the NOVA1 gene.

Description

Method for specifically modifying monkey-derived NOVA1 gene into human gene

Technical Field

The invention belongs to the fields of genetic engineering and genetic modification, and in particular relates to a preparation method of a vector for specifically modifying a monkey source NOVA1 gene into a human source gene by a single base editing technology.

Background

Human evolution is generally considered a long-lasting behavior, which is continued due to genetic mutations and natural selection. Gene mutations are occasional in humans because parents inherit genes carrying certain specific traits to offspring. These gene transfers may occur through natural selection, with carriers of dominant genes being better able to survive, adapt to the environment, and reproduce the offspring.

Niander is a close relative to the ancestor of modern Europe, beginning 12 years ago, and they predominate throughout Europe, western Asia, and North Africa, but by twenty-four thousands of years ago, these ancient humans disappeared. The 2021 Muotri team found 61 different genes by analyzing the differences between genomes from modern humans and niandert, with NOVA1 being the major gene regulator, affecting splicing of other genes during brain development. During the biological evolution process, the base encoding amino acid 200 of the NOVA1 gene is gradually subjected to gene mutation, and the isoleucine mutation encoded by ATA is changed into valine encoded by GTA. Muotri team uses CRISPR gene editing technology to edit modern human stem cells, introduces variation in the Niandela human NOVA1 gene, and simulates "Niandela humanized" brain organ. Some differences in the development of modern and niandde humanized brains were found. It would be of great reference to the study of biological evolution if a relatively low version of organisms, and in particular primate organisms such as monkeys, gorillas, etc. having a similar degree of evolution to humans could be modified to a human version. However, this part of the work is not reported at present.

Genome editing (simply called gene editing) technology is a genetic engineering technology for performing targeted modification on genome by utilizing artificial nuclease, and is a research hotspot in the field of life science at present. The CRISPR/Cas9 gene editing system improves the efficiency of gene knockout and site-directed modification (including site-directed mutation, gene insertion and the like), but the efficiency of gene site-directed mutation based on a homologous recombination mechanism is still low. To increase the efficiency of site-directed mutagenesis, a single base editing system that binds CRISPR/Cas9 and cytosine deaminase was reported sequentially. The single base editing technology can replace single base of DNA without double strand break, so that it has high efficiency and accurate gene editing capacity, and may be used in introducing point mutation into animal and plant cell for cultivating gene editing animal and plant with ideal phenotype and correcting pathogenic point mutation for gene treatment of genetic disease. The adenine single base editor (ABE) can realize efficient single base conversion from adenine (A) to guanine (G) at a target site, and can effectively modify an ATA version of monkey genome into a GTA version of humanized genome.

Disclosure of Invention

The invention aims to provide a novel method for carrying out single-point directional transformation on monkey genome NOVA1 genes.

In the research paper of science in 2021, muotri team uses CRISPR gene editing technology to edit stem cells of modern human beings, and introduces variation in the Niandela human NOVA1 gene, simulating "Niandela humanized" brain organ. The patent goes against the way, and in the monkey-source cell cos7 (the cells of the Niandert are impossible to obtain, and cos7 is a common tool cell of the monkey source and is a model cell for the experiment of the monkey-source cell), the monkey-source gene template is modified into the human-source gene template, and a model of technical possibility is provided for the evolution of follow-up monkeys, chimpanzees, ancient mankind and the like for the research of modern mankind. The most effective and straightforward way is to modify the gene directly on the monkey, obtain a personnel version of the monkey, and conduct a study of brain development on animal individuals. However, the present invention does not develop such experiments under such experimental conditions.

The invention provides a preparation method of a vector containing a monkey-source NOVA1 gene specifically modified into a vector of a human-source gene by a single-base editing technology. The single base editing technique can introduce point mutations with high efficiency without generating double strand breaks, but not all gene mutations can be realized by using the single base editing technique, nor all homologous gene changes can be realized by using the single base editing technique. The research of the invention discovers that the first A base in the 200 th amino acid (ATA) of the monkey source NOVA1 gene perfectly meets the requirement of single base editing, not only is the most suitable single base editing window, but also the single base change can generate a remarkable phenotype. On the basis of comprehensively considering the feasibility of a single base editing technology in scientific logic and the value of clinical research, the invention can modify isoleucine of a No. 200 site of NOVA1 in monkey-source cells into valine coded by GTA through the single base editing technology, and obtain specific and stable site-directed mutagenesis materials.

In one aspect, the invention provides a method for modifying a monkey-derived NOVA1 gene into a human gene, comprising the steps of:

determining a specific site for modifying a monkey cell NOVA1 genome into a human gene and the sgRNA, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1;

the combined sequence of U6-sgRNA-scafaold containing the sgRNA is synthesized, the puromycin-containing fragment is amplified, and the puromycin-containing fragment is constructed on an original vector pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP (RTW 5025) to become a final vector pCMV-T7-ABEmax (7.10) -NOVA1.

Preferably, the method comprises: the monkey-derived cells cos7 were resuscitated and cultured prior to transfection of the constructed pCMV-T7-ABEmax (7.10) -NOVA1 into cos7 cells.

Preferably, the specific site of monkey cell NOVA1 genome modification to a human gene and the sgRNA are determined by screening the specific site of monkey cell NOVA1 genome modification to a human gene and the sgRNA.

Preferably, identification or confirmation of the final vector constructed is capable of modifying the monkey-derived genomic NOVA 1-specific site to a human-specific sequence after transfection of the constructed pCMV-T7-ABEmax (7.10) -NOVA1 into cos7 cells.

Preferably, identifying or confirming that the constructed final vector is capable of modifying the monkey genome NOVA 1-specific site to a human-specific sequence means that the constructed pCMV-T7-ABEmax (7.10) -NOVA1 is transfected into cos7 cells or 293T cells, and the constructed final vector is confirmed to be capable of modifying the monkey genome NOVA 1-specific site to a human-specific sequence by puromycin screening and genome identification.

Preferably, the combined sequence is shown as SEQ ID NO.2, the sequence of the amplified fragment is shown as SEQ ID NO.3, the sequence of the original vector is shown as SEQ ID NO.4, and the sequence of the final vector is shown as SEQ ID NO. 5.

In a second aspect, the invention also provides the use of the above method comprising modifying a monkey-derived NOVA1 gene to a human gene using the method.

According to some embodiments of the invention, the specific modification of the monkey-derived NOVA1 gene to a functional human gene is achieved based on single base editing techniques. The invention uses single base editing technology, realizes site-directed mutation of No. 200 amino acid of NOVA1 gene on the basis of not cutting genome, and provides a possible technical means and clinical thought for the subsequent study of specific sites of NOVA1 gene. Therefore, the invention has a certain scientific research value and practical significance in innovation and practicability.

Preferably, the method can be used to construct vectors specifically modifying monkey genome NOVA1 to a human gene.

In a third aspect, the invention provides a vector prepared by the method, wherein the monkey genome NOVA1 in the vector is specifically modified into a human gene.

Preferably, the vector is a puromycin-containing fragment in the combined sequence of the amplified U6-sgRNA-scafaold, and is constructed on the original vector pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP (RTW 5025) to obtain the final vector pCMV-T7-ABEmax (7.10) -NOVA1.

In a fourth aspect, the invention provides a cell prepared from the vector prepared by the method, wherein the monkey genome NOVA1 in the cell is specifically modified to a human gene.

Preferably, the cell is a cos7 cell line or a 293T cell line.

In a preferred embodiment of the present invention, there is provided a method for preparing a vector for specifically modifying a monkey-derived NOVA1 gene into a human gene by a single base editing technique, comprising the steps of:

s1, resuscitating, culturing and identifying a base sequence of a specific site of a genome NOVA1 in a gene of monkey cell cos7;

s2, designing a sgRNA-based base sequence aiming at a monkey-derived cell NOVA1 genome specific site, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1;

s3, synthesizing a U6-sgRNA-scafaold combined sequence containing the sgRNA of S2, amplifying a puromycin-containing fragment, and constructing an original vector pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP (RTW 5025) to form a final vector named pCMV-T7-ABEmax (7.10) -NOVA1. The combined sequence, the amplified fragment, the original vector sequence and the final vector sequence are shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO. 5;

s4, the pCMV-T7-ABEmax (7.10) -NOVA1 constructed in the S3 is transfected into cos7, and the constructed final vector is confirmed to be capable of modifying the monkey-derived genome NOVA1 specific site into a human-derived specific sequence through puromycin screening and genome identification.

The above-mentioned sequences involved are:

SEQ ID NO.1：5`-AAGGCTGTAATGGAGCAGTC-3`；

SEQ ID NO.2：

SEQ ID NO.3：

SEQ ID NO.4：

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SEQ ID NO.5：

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the research published by the Muotri team in 2021 on the national top journal science shows that NOVA1 is the major gene regulator and can affect splicing of other genes in the brain development process. Therefore, the invention carries out specific mutation on the amino acid of the No. 200 locus of the NOVA1 gene, and tests show that the vector and the cell prepared by the invention have no risk of off-target and risk of non-specific editing possible by the CRISPR/Cas9 technology, and have important research value.

The invention has at least the following beneficial effects:

1. according to the invention, the specific site and the sgRNA of the monkey cell NOVA1 genome modified into the human gene are determined by screening the specific site and the sgRNA of the monkey cell NOVA1 genome modified into the human gene, and the directed mutation of No. 200 amino acid of the NOVA1 gene is realized on the basis of no need of genome cutting by using a single base editing technology.

2. The invention constructs the vector and the cell for modifying the monkey cell NOVA1 genome into the human gene, which is favorable for stably preserving the material for editing the specific gene.

3. The invention constructs a simian cell strain, provides a technical means for deeply researching the function of the gene, and also provides a clinical idea for researching a characteristic disease (based on a disease caused by single base mutation).

Drawings

In order to more clearly illustrate the technical solutions of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that, for some embodiments of the present application, each drawing in the following description may be further obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a diagram showing the sequencing peaks of NOVA1 at amino acid position 200 in cos7 cells and 293T cells according to an embodiment of the present invention.

FIG. 2 is an electrophoretogram of a primary vector constructed in the examples of the present invention, which was subjected to double cleavage using MluI and SpeI enzymes.

FIG. 3 is a graph showing the effect of transfection of cos7 cells with pCMV-T7-ABEmax (7.10) -NOVA1 plasmid in the examples of the present invention.

FIG. 4 is a diagram showing the sequencing peaks of NOVA1 at amino acid position 200 in cos7-NOVA1 cells according to an embodiment of the present invention.

Detailed Description

The invention discloses a preparation method and application of a vector for specifically modifying a monkey-source NOVA1 gene into a human gene by a single base editing technology, wherein a CRISPR/Cas9 system is used for introducing a Cas9 protein expression gene, a deoxyadenosine deaminase gene, a puromycin resistance gene and a fluorescent marker gene into a tool cell cos7 cell, positive clone cell strains are screened by resistance and fluorescence, and then genome PCR identification is carried out, so that the constructed vector can be determined to effectively directionally modify the monkey-source NOVA1 gene into a human gene sequence. The cell strain constructed by the invention is constructed based on a single-base gene editing system, does not cause genome rupture, and is a tool cell strain capable of stably inheriting.

The technical solutions will be clearly and completely described below by means of embodiments of the present application, it being apparent that the described embodiments are only some of the preferred embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the inventors, are within the scope of the present application based on the embodiments herein.

EXAMPLE 1 cultivation and identification of the NOVA1 Gene of simian cells cos7 and human cells 293T

1.1 culturing cos7 cells and 293T cells

Cos7 cells and 293T were recovered and cultured. The formulation of the cell culture medium is shown in table 1 below:

TABLE 1 Cos7 cell complete Medium composition Table

1.2 NOVA1 Gene identification in cos7 cells and 293T cells

Cultured cos7 cells and 293T cells were harvested and subjected to genome extraction using a genome extraction kit (purchased from tenna, cat No. DP 304).

Genomic PCR amplification primers were designed for the I200 gene of the NOVA1 gene based on the monkey gene NOVA1 sequence given on the NCBI website. The designed primer sequences were as follows:

NOVA1-GF1：5`-GGTAAAGATTATAGTTCCCAACAGC-3`,SEQ ID NO 6；

NOVA1-GR1：5`-TCTTCTGGATGATAAGTTCAACAGC-3`,SEQ ID NO 7；

amplified genomic fragments were sequenced using NOVA1-GF 1. The sequencing results are shown in FIG. 1, in which amino acid number 200 of the monkey cos7 cell NOVA1 gene is ATA encoded isoleucine, and amino acid number 200 of the human 293T cell NOVA1 gene is GTA encoded valine.

Example 2 construction of a plasmid suitable for Single base editing targeting monkey-derived NOVA1 genome I200

2.1 design of sgRNA for monkey-derived NOVA1 genome I200

According to the monkey-derived NOVA1 gene sequence given on NCBI website, the sgRNA of I200 of the targeted NOVA1 gene is designed, and the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 1.

2.2 Synthesis of the U6-sgRNA-scafaold Combined sequence (SEQ ID NO. 2) and addition of MluI and SpeI cleavage sites on both sides.

2.3 the original vector pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP (RTW 5025) (purchased from vast Ling Bio, cat. No. P20294, SEQ ID NO. 4) was double digested with MluI and SpeI enzymes. The enzyme digestion effect is shown in figure 2;

2.4 ligation of the fragment synthesized in 2.2 with the 2.3 digested plasmid fragment to obtain vector named pCMV-T7-ABEmax (7.10) -NOVA1-P2A-EGFP (SEQ ID NO. 4).

2.5 double digestion of pCMV-T7-ABEmax (7.10) -NOVA1-P2A-EGFP constructed in 2.4 with XbaI and KpnI was performed and a combined fragment PGK promtor-puroR-WPRE fragment containing puromycin resistance selection marker (sequence shown as SEQ ID NO. 5) was ligated to form the final vector containing both fluorescence and resistance selection markers, designated pCMV-T7-ABEmax (7.10) -NOVA1 (sequence shown as SEQ ID NO. 5).

Example 3 cell transfection, screening and identification of the obtained genetically modified cell lines

3.1 cell transfection

The invention adopts a Hantao transfection reagent (product number: HB-TRLF-1000) and carries out transfection according to the steps of a product specification. The third day after transfection (72 hours later), the proportion of cells with green fluorescence was observed, and the more cells positive for the proportion of green fluorescence in the microscope field, the stronger the green fluorescence, indicating higher transfection efficiency, and the experimental results are shown in FIG. 3.

3.2 puromycin screening of positive cells

Cell selection was performed using cells transfected with 3.1 cells, with puromycin added to normal cos7 cell complete medium (see Table 1). The puromycin screening concentration used in the invention is 2 mug/ml, and the screening time is 3 days. After 3 days of screening, the cells were passaged and replaced with normal cos7 cell complete medium, and after 4 days the samples were collected for subsequent testing. Cells transfected to work pCMV-T7-ABEmax (7.10) -NOVA1, designated cos7-NOVA1, can be enriched by puromycin selection.

3.3 genome identification

To identify cos7-NOVA1 as cells in which the monkey-derived NOVA1 gene was specifically modified to a functional human gene by single base editing techniques, the cultured cos7-NOVA1 cells were harvested and subjected to genome extraction using a genome extraction kit (purchased from Tiangen organism, accession number DP 304).

Genomic PCR amplification primers were designed for the I200 gene of the NOVA1 gene based on the monkey gene NOVA1 sequence given on the NCBI website. The designed primer sequences were as follows:

NOVA1-GF1：5`-GGTAAAGATTATAGTTCCCAACAGC-3`，SEQ ID NO.6；

NOVA1-GR1：5`-TCTTCTGGATGATAAGTTCAACAGC-3`，SEQ ID NO.7。

the amplified genomic fragment was sequenced using NOVA1-GR 1. The sequencing results are shown in FIG. 4, in which amino acid number 200 of the NOVA1 gene of cos7-NOVA1 cells contains both monkey-derived ATA encoded isoleucine and humanized GTA encoded valine. Illustrating that a vector specifically modifying monkey-derived NOVA1 genes into functional human genes by single base editing technique constructed in the present invention is capable of modifying the I200 orientation of monkey-derived NOVA1 genes into V200.

In conclusion, the invention uses a single base editing technology, realizes site-directed mutation of No. 200 amino acid of the NOVA1 gene on the basis of not cutting genome, and provides a possible technical means and clinical thought for later research on specific sites of the NOVA1 gene. Therefore, the invention has a certain scientific research value and practical significance in innovation and practicability.

The above-described embodiments are merely specific embodiments of the present application, but the scope of protection of the present application is not limited thereto, and any changes or substitutions that can be suggested by one skilled in the art without creative efforts are intended to be included in the scope of protection of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims in the present application.

Claims (10)

1. A method for modifying a monkey-derived NOVA1 gene to a human gene comprising the steps of:

determining a specific site for modifying a monkey cell NOVA1 genome into a human gene and the sgRNA, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1;

the combined sequence of U6-sgRNA-scafaold containing the sgRNA is synthesized, the puromycin-containing fragment is amplified, and the puromycin-containing fragment is constructed on an original vector pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP (RTW 5025) to become a final vector pCMV-T7-ABEmax (7.10) -NOVA1.

2. The method of claim 1, wherein the method comprises:

recovering and culturing monkey-derived cells cos7 prior to transfection of the constructed pCMV-T7-ABEmax (7.10) -NOVA1 into cos7 cells;

determining the specific site and the sgRNA of the monkey cell NOVA1 genome modified into the human gene by screening the specific site and the sgRNA of the monkey cell NOVA1 genome modified into the human gene; or alternatively

After transfection of the constructed pCMV-T7-ABEmax (7.10) -NOVA1 into cos7 cells, identification or validation of the constructed final vector enabled modification of monkey genome NOVA 1-specific sites to human-specific sequences.

3. The method of claim 2, wherein,

identification or confirmation of the constructed final vector the monkey genome NOVA1 specific site could be modified to a human specific sequence by transfecting the constructed pCMV-T7-ABEmax (7.10) -NOVA1 into cos7 cells or 293T cells and confirming the constructed final vector could modify the monkey genome NOVA1 specific site to a human specific sequence by puromycin selection and genome identification.

4. The method of claim 1, wherein,

the combined sequence is shown as SEQ ID NO. 2;

the sequence of the amplified fragment is shown as SEQ ID NO. 3;

the sequence of the original vector is shown as SEQ ID NO. 4; or alternatively

The sequence of the final vector is shown in SEQ ID NO. 5.

5. The method of claim 1, wherein,

the steps of synthesizing the U6-sgRNA-scafaold combined sequence containing the sgRNA, amplifying the puromycin-containing fragment, and constructing the puromycin-containing fragment on an original vector pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP to form a final vector pCMV-T7-ABEmax (7.10) -NOVA1 comprise:

s2-1, designing sgRNA of I200 of a targeted NOVA1 gene according to a monkey-derived NOVA1 gene sequence, wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO.1;

s2-2, synthesizing a combined sequence of U6-sgRNA-scafaold, and adding MluI and SpeI enzyme cutting sites on two sides respectively;

the original vector pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP was double digested with MluI and SpeI enzymes;

s2-3, connecting the fragment synthesized by the S2-2 with the plasmid fragment cut by the S2-3 to obtain a vector named pCMV-T7-ABEmax (7.10) -NOVA1-P2A-EGFP;

s2-4, carrying out double digestion on pCMV-T7-ABEmax (7.10) -NOVA1-P2A-EGFP constructed in S2-3 by XbaI and KpnI, and connecting the two digested fragments into a combined fragment PGK promor-puroR-WPRE fragment containing puromycin resistance screening marker to form a final vector containing both fluorescent screening and resistance screening marker, and naming the final vector as pCMV-T7-ABEmax (7.10) -NOVA1.

6. Use of the method of claim 1, wherein the method is used to construct a variant specifically modifying monkey genome NOVA1 to a human gene.

7. The vector prepared by the method of claim 1, wherein the monkey genome NOVA1 in the vector is specifically modified to a human gene.

8. The vector of claim 7, wherein the vector is a puromycin-containing fragment of the combined sequence of U6-sgRNA-scafaold amplified and constructed to the original vector

The final vector pCMV-T7-ABEmax (7.10) -NOVA1 was obtained on pCMV-T7-ABEmax (7.10) -SpRY-P2A-EGFP (RTW 5025).

9. A cell comprising the vector of any one of claims 7 to 8, wherein the monkey genome NOVA1 in the cell is specifically modified to a human gene.

10. The cell of claim 9, wherein the cell is a cos7 cell line or a 293T cell line.

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