CN108546703B

CN108546703B - Four sgRNAs designed for human ADRB2 gene

Info

Publication number: CN108546703B
Application number: CN201810382911.3A
Authority: CN
Inventors: 施明; 郑骏年; 刘丹; 孙毓
Original assignee: Xuzhou Medical University
Current assignee: Jiangsu Jicui Juchuang Biotechnology Research Institute Co ltd
Priority date: 2018-04-26
Filing date: 2018-04-26
Publication date: 2021-04-13
Anticipated expiration: 2038-04-26
Also published as: CN108546703A

Abstract

The invention belongs to the technical field of gene modification, and discloses four sgRNA sequences designed for human ADRB2 genes. The CDS sequence of the human ADRB2 gene is searched by logging in a website, and four sgRNAs are designed by adopting a head-to-head arrangement mode at the end close to 5' of the CDS region according to the design principle of the sgRNAs. On the basis, a single sgRNA expression plasmid and a multiple sgRNA vector are respectively constructed. T7EN1 enzyme digestion identification and TA clone sequencing prove that after HEK293T cells are transfected by two plasmids, the Cas9 protein can be guided to cut a target gene, and particularly the gene modification efficiency of the multiple sgRNAs can reach 100%.

Description

Four sgRNAs designed for human ADRB2 gene

Technical Field

The invention belongs to the technical field of CRISPR/Cas9 gene editing, and particularly relates to four sgRNAs with pointers designed for human ADRB2 genes.

Background

The gene editing technology is a technology which has been developed in recent years and can precisely modify a gene. In the field of scientific research, gene editing technology can be used for rapid construction of model organisms; in the agricultural field, the technology can be used for plant variety modification; in the field of health care, the technology can also achieve the purpose of treating diseases by modifying human self genes. Therefore, the gene editing technology has extremely wide development prospect and application value.

At present, the gene editing technology mainly comprises Zinc Finger Nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR/Cas 9). In principle, three gene editing techniques are all to cause genome fragmentation through targeted recognition and cleavage of specific sequences, and achieve the purpose of precise modification of genes in the process of non-homologous end joining repair (NHEJ) and homologous recombination repair (HR) of cells.

The zinc finger nuclease is used as a first generation gene editing technology, and the targeted operation on the gene is realized for the first time. The principle is as follows: zinc finger ribonucleases consist of a DNA recognition domain and an endonuclease. The DNA recognition domain is composed of a series of zinc finger protein modules which are connected in series, and each zinc finger protein module recognizes and combines a specific triplet base, thereby realizing the targeted cutting of the endonuclease to a specific locus of the genome. Although the gene targeting efficiency of zinc finger nuclease can reach 30% in the literature, the development of the zinc finger nuclease still has great limitation. On one hand, context sequence dependency exists in the recognition domain of the zinc finger nuclease, so that the design and screening difficulty is increased; on the other hand, off-target cleavage of zinc finger nucleases can cause cytotoxicity, and the application of the zinc finger nucleases in the field of gene therapy is restricted.

Transcription activator-like effector nucleases (TALENs) are the second generation gene editing technology, and TALENs are simpler in design and more flexible in use than ZFNs. TALENs act on a similar principle to ZFNs, but specifically recognize DNA sequences as TALEN proteins. The TALEN protein utilizes the conservative and constant corresponding relation formed by A, G, C, T based on the 12/13 th amino acid residue in the repeating unit of the TALEN protein, thereby realizing the recognition and the cutting of any specific site on the genome.

CRISPR/Cas9 is a newly developed third generation gene editing technology that is derived from the adaptive immune system of bacteria and archaea. The first two generations of gene editing technologies are different, and the CRISPR/Cas9 system is used for identifying and cutting a specific DNA sequence by using RNA-mediated Cas9 endonuclease so as to accurately edit a genome. Among them, an RNA that functions as a guide is called sgRNA (single-guide RNA). The CRISPR RNA is combined with a target sequence through a base complementary pairing principle to play a role in accurate positioning, and the tracrRNA (trans-acting CRISPR RNA) is used for assisting a crRNA/Cas9 protein to form a stable compound to realize accurate positioning of Cas9 on a genome, further shearing a DNA double strand to form a DSB (double Stranded breaks) notch, activating a DNA damage repair mechanism of a cell, causing a frameshift mutation in the repair process, and finally realizing the knockout of a target gene.

Once appeared, the CRISPR/Cas9 system is widely applied in the basic research field and research and development field, mainly because it has incomparable advantages with the first two generations of gene editing technology. Firstly, the construction process is simple, the operability is strong, and the cost is low. Secondly, the simultaneous knockout of single gene and multiple sites or multiple genes can be realized, and the gene editing efficiency is greatly improved. Thirdly, the plasmid vector has small volume, and the cytotoxicity in the transfection process is reduced. Finally, the occurrence frequency of the editable sites on the genome is high, and the proper sites are easy to select for efficient gene editing operation. Besides, compared with the gene interference technology (RNAi) which is most widely applied in the field of gene silencing at present, the CRISPR/Cas9 has the same advantages that:

disclosure of Invention

According to the design principle of sgRNAs, four sgRNAs are designed at the end close to 5' of a CDS (protein coding region) of a humanized beta 2-AR (ADRB2) gene in a head-to-head arrangement mode, and single sgRNA recombinant plasmids and multiple sgRNA recombinant plasmids constructed by orderly connecting the 4 sgRNAs in series are respectively constructed. T7EN1 enzyme digestion and TA clone sequencing prove that effective cutting of ADRB2 gene can be realized by single sgRNA or multiple sgRNAs obtained after 4 sgRNAs are connected in series, but the cutting activity of the multiple sgRNAs is the highest, and 100% editing efficiency can be achieved.

The technical scheme of the invention is as follows: the CDS sequence of the human ADRB2 gene was retrieved by logging into the UCSC Genome Browser Home website. According to the design principle of sgrnas, four sgrnas are designed in a head-to-head arrangement near the 5' end of the CDS region (protein coding region). The sequences of the sgRNA1, the sgRNA2, the sgRNA3 and the sgRNA4 are as follows:

sgRNA1：5’-CCTGCGTGACGTCGTGGTC-3’，

sgRNA2：5’-GTCATGTCTCTCATCGTCC-3’，

sgRNA3：5'-ATCCACTGCGATCACGCAC-3'，

sgRNA4：5'-AGCCTGCTGACCAAGAATA-3'，

and connecting the synthesized 4 sgRNAs with pGL3-U6-sgRNA-PGK-puromycin vectors respectively to construct 4 single sgRNA recombinant plasmids targeting ADRB2 genes. Through the enzyme digestion identification of T7EN1, 4 sgRNAs can realize the specific cleavage of ADRB2 gene.

The 4 sgRNAs are further orderly connected in series and connected with a eukaryotic expression vector pGL3-U6-sgRNA-ccdB-EF1 alpha-Puro to form a multiple sgRNA recombinant plasmid of a targeted ADRB2 gene. The detection result of the T7EN1 enzyme digestion shows that the multiple sgRNAs can obtain higher cleavage activity compared with the single sgRNA. The sequencing results of the TA clone also confirmed that the editing efficiency of multiple sgrnas against specific target sites of ADRB2 gene was as high as 100%.

The invention has the following beneficial effects: according to the invention, 4 sgRNAs designed aiming at a CDS region at the 5' end of the human ADRB2 gene can obtain higher cutting activity after being singly or orderly connected in series, so that a DNA repair mechanism of a cell is activated, the deletion or insertion of a base is caused, the frameshift mutation is finally triggered, and the high-efficiency knockout of the ADRB2 gene is realized. Moreover, multiple sgrnas constructed by orderly connecting 4 sgrnas in series in a head-to-head manner can more accurately and efficiently guide Cas9 protein to cleave DNA double strands, further reduce off-target effects, and finally successfully knock out the ADRB2 gene in cells.

Drawings

FIG. 1 shows the positions of four sgRNAs designed according to the present invention in the CDS region of human ADRB2 gene;

FIG. 2 shows the results of enzyme digestion detection of four single sgRNA expression plasmids T7EN 1;

fig. 3 shows the results of enzyme digestion detection of the multiple sgRNA recombinant plasmid T7EN 1;

FIG. 4 is a schematic diagram showing the sequencing results of the 1# TA clone;

FIG. 5 is a schematic diagram showing the sequencing results of 2# TA clone;

FIG. 6 is a schematic diagram showing the sequencing results of the 3# TA clone;

FIG. 7 is a schematic diagram showing the sequencing results of # 4 TA clone;

FIG. 8 is a schematic diagram showing the sequencing results of 5# TA clone;

FIG. 9 is a diagram showing the sequencing results of the 6# TA clone;

FIG. 10 is a diagram showing the sequencing results of the 7# TA clone;

FIG. 11 is a diagram showing the sequencing results of # 8 TA clone;

FIG. 12 is a diagram showing the sequencing results of # 9 TA clone;

FIG. 13 is a diagram showing the sequencing results of the 10# TA clone.

Detailed Description

In the following, the technical solutions of the present invention will be further described in detail, and the parts of the present invention that are not described in detail adopt the existing mature technologies.

The present invention will be described in further detail with reference to the accompanying drawings.

The present invention is directed to four sgrnas designed for the human ADRB2 gene. First, the CDS sequence of human ADRB2 gene was searched by registering the UCSC Genome Browser Home website. Then, according to the design principle of sgrnas, 4 target sites were selected and four sgrnas were designed near the 5' end of the CDS region by head-to-head design (see fig. 1). A single sgRNA expression vector targeting the ADRB2 gene is constructed by connecting the single sgRNA expression vector with pGL3-U6-sgRNA-PGK-puromycin vector, and T7EN1 enzyme digestion identification proves that 4 sgRNAs can effectively guide Cas9 endonuclease to cut a specific site of a target gene (as shown in figure 2). Finally, 4 sgrnas are orderly connected in series and are connected with pGL3-U6-sgRNA-ccdB-EF1 alpha-Puro, so that a multiple sgRNA recombinant plasmid is constructed and transfected into an HEK293T cell. The T7EN1 enzyme digestion result shows that the multiple sgRNA recombinant plasmid has higher cleavage activity than the single sgRNA plasmid (see fig. 3). TA clone sequencing proves that the knockout efficiency of the multiple sgRNAs on the target gene is up to 100%, and various gene mutation types including deletion and insertion of bases, deletion and rearrangement of large fragments (as shown in figures 4-13) appear. Thus, it is confirmed that the 4 sgrnas targeting ADRB2 gene designed by us can realize high-efficiency knockout of target gene by single or combined application.

The designed 4 sgrnas were named: sgRNA1, sgRNA2, sgRNA3, and sgRNA 4. The specific sequence is as follows:

sgRNA1：5’-CCTGCGTGACGTCGTGGTC-3’，

sgRNA2：5’-GTCATGTCTCTCATCGTCC-3’，

sgRNA3：5'-ATCCACTGCGATCACGCAC-3'，

sgRNA4：5'-AGCCTGCTGACCAAGAATA-3'，

the present invention and its embodiments have been described above, and the description is not intended to be limiting, and what is shown in the drawings is only one embodiment of the present invention, and the actual solution is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. The sgRNA designed for human ADRB2 gene consists of sgRNA1, sgRNA2, sgRNA3 and sgRNA4, and the corresponding nucleotide sequences of the sgRNA1, the sgRNA2, the sgRNA3 and the sgRNA4 are as follows:

sgRNA1：5’-CCTGCGTGACGTCGTGGTC-3’，

sgRNA2：5’-GTCATGTCTCTCATCGTCC-3’，

sgRNA3：5'-ATCCACTGCGATCACGCAC-3'，

sgRNA4：5'-AGCCTGCTGACCAAGAATA-3'，

the sgRNA is characterized in that sgRNA1, sgRNA2, sgRNA3 and sgRNA4 are connected in series to form the sgRNA.

2. Use of the sgRNA of claim 1 to guide cleavage of the ADRB2 gene by a Cas9 endonuclease.

3. The use of claim 2, wherein the sgRNA directs Cas9 endonuclease cleavage of the ADRB2 gene resulting in deletion or insertion of bases.

4. The use of claim 2, wherein the sgRNA directs Cas9 endonuclease to cleave the ADRB2 gene resulting in gene knock-out.

5. An expression vector comprising the sgRNA of claim 1.