LU102620B1 - A gRNA and Related Kit and Application - Google Patents

Abstract

The invention discloses a gRNA and related kit and application. Specifically, the nucleotide sequence of the gRNA comprises one or two sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2. The invention further discloses biological materials and kits related to the gRNA. Through the provided gRNA, the present invention can realize the modification of the CDK13 gene in the mouse genome based on CRISPR-Cas9 technology, which is of great significance for constructing a CDK13 gene-modified mouse model.

Description

Description LU102620 A gRNA and Related Kit and Application

TECHNICAL FIELD The invention relates to a gRNA and kit and application thereof, belonging to the technical field of genetic engineering.

BACKGROUND CRISPR/Cas system (Clustered Regularly Interspaced Shot Palindromic Repeats/CRISPR- associated system), which is derived from bacterial acquired immunity, is a targeted modification of target genes by RNA-mediated Cas protein. After successfully modifying mammalian cells in 2013, the Type II CRISPR/Cas9 system modified by researchers has now been applied to genetic modification of various model organisms. The vector construction of CRISPR/Cas9 system is simple, fast, easy to operate, timesaving, labour-saving and short in cycle, plus it is suitable for almost all species. For each gene, CRISPR/Cas9 only needs to construct a gRNA (single guideRNA), which is highly efficient and has little restriction on sequence selection. Besides, it only needs GG to appear on the genome. Compared with zinc- finger nucleases (AFNs) and TALEN, CRISPR/Cas9 system has the same or higher genetic modification efficiency and is much cheaper, but it also has higher off-target effect than that of TALEN. However, the use of paired truncated gRNA or FoKI-dCas9 by gRNA/Cas9-D10A can greatly reduce off-target effects. CDK 13 (cyclin-dependent kinases 13) is the latest member of the cell cycle-dependent kinase family, which has a 20 ATP-dependent serine-threonine protein kinase structure at the C- terminal. By integrating signals inside and outside the cell, it can realize the regulatory function of cell cycle and gene transcription process. CDK12 and CDK13 have many homologous sequences, which may play an important role in RNA transcription and processing. Human CDK13 protein has a large molecular weight of 165 kDa, and it can combine with Cyclin K to form a protein complex that can perform biological functions. CDK13 hyperphosphorylated LU102620 serine protease Omi/HtrA2 can promote apoptosis. At present, there is no animal model modified by CDK13. Therefore, based on above-mentioned functions of CDK13, it is of great significance to construct animal model by CRISPR-Cas9 system for studying its related functions.

SUMMARY The purpose of the present invention is to provide a gRNA and related kit and application to solve above problems existing in the prior art. By providing gRNA that can specifically recognize the CDK 13 gene, as well as gRNA-related biological materials and kits, the invention can modify the CDK13 gene of mouse genome, which is of great significance for the construction of CDK 13 gene-modified mouse models. To achieve the above purpose, the present invention provides the following scheme. The invention provides a gRNA, which is responsible for identifying the nucleotide sequence of the target gene CDK13 fragment. Specifically, the nucleotide sequence of gRNA comprises one or two sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2. The invention also provides biological materials related to the gRNA, including (1) a DNA molecule encoding the gRNA of Claim 1; (2) an expression cassette containing the DNA molecule in (1); (3) expression vectors composed of DNA molecules in (1) or expression cassettes in (2). Preferably, the nucleotide sequence of the DNA molecule is composed of one or two sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4. Preferably, the expression cassette further comprises DNA molecules that can encode Cas9. A kit is further disclosed by the invention, which includes the gRNA or biological materials. Moreover, the kit contains matched detection reagent, which is composed of the following components: template 1uL, reaction buffer 2.5uL, upstream primer/downstream primer 0.5uL,

Mg” 2.5uL, dNTPs 0.5uL and Taq DNA polymerase 0.25uL. Finally, ddH2O is supplemented LU102620 to 20uL. The invention discloses the following technical effects. The gRNA that can specifically recognize the CDK13 gene provided by the present invention is responsible for recognizing the target fragment region, and its nucleotide sequence is one or two of SEQ ID NO: 1 - SEQ ID NO: 2. On the one hand, the gRNA can recognize the nucleotide sequence of the CDK13 fragment region. On the other hand, through the mediation of gRNA, the present invention can realize the modification of CDK13 gene, which is of great significance for constructing CDK13 gene-modified mouse model.

DESCRIPTION OF THE INVENTION The technical scheme of the present invention will be explained in detail with following specific embodiments. Moreover, it should not be regarded as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics and embodiments of the present invention. Embodiment 1 Construction of Cas9/gRNA expression vector and activity detection of gRNA. The Cas9/gRNA expression vector is constructed for specific recognition of CDK13 gene target sites. 1) Based on the analysis of CDK13 gene, selecting an appropriate fragment region and designing a pair of corresponding gRNA sequences according to the target gene. Then synthesizing gRNA sequences as follows. 2) Construction of gRNA targeting vector.

According to the two gRNA sequences in 1), they were ligated into Bsal enzyme digestion LU102620 pUC57 vector. 3) The constructed pUC57-gRNA vector was water-bathed at 37°C for 1h, and then subjected to 2% agarose electrophoresis.

The enzyme digestion product was recovered.

And then annealing the gRNA primer.

Finally, connecting the annealed product and the recovered enzyme digestion product, and using it to transform Escherichia coli.

Selecting a monoclonal antibody for PCR, and the PCR result was positive and sent to sequencing for verification.

The amplification primers are as follows: Upstream primer: 5’-CAAATACAGCAAGCACCTG-3°. Downstream primer: 5’-AGTTCTTCCTCAGTTGTAACC-3". PCR reaction system:

Reagent Dosage

Template Tul

Reaction buffer 2.5uL

Upstream primer 0.5uL for each

/downstream primer

Mg” 2.5uL dNTPs 0.5uL

Taq DNA polymerase 0.25uL ddH:0 Supplement to 20uL The PCR amplification procedure followed the cycling protocol: denaturation at 95°C for Smin, denaturation at 95°C for 30s, annealing at 58°C for 30s, extension at 72°C for 30s, totalling 30 cycles; and finally extension at 72°C for Smin, maintaining 4°C.

The results showed that there was obvious peak noise in the target site sequencing peaks of gRNA1 and gRNA2 samples, indicating their activity.

At the same time, it was found that the amplified products of gRNA1, gRNA2 and gRNA1-gRNA showed obvious bright bands after enzyme digestion, which indicated that gRNA1 and gRNA2 caused sequence mutation in the target area.

The cutting efficiency of gRNA1 and gRNA2 reached about 25% by calculating the LU102620 band brightness with Image software.

The above results indicate that gRNA1 and gRNA-mediated Cas9 protein have better modification effect on CDK13. Embodiment 2 Screening of mouse neural stem cell lines with targeted knockout of CDK13 gene.

The cultured pregnant mice were killed with broken neck, and the mouse neural stem cells were obtained by referring to Isolation, Culture and Identification of C57 Mouse Neural Stem Cells by Wan Li et al.

On the day before transfection, mouse neural stem cells were resuscitated in a 6cm plate, and when the cells reached 70%-80% confluence, the cells could be electrically transfected.

The specific operation is as follows. a.

Collecting cells and adjusting the number of cells to 1x10%tube.

Then centrifuging cells at 50rpm for 5min so as to remove the culture solution as clean as possible. b.

Adding 100uL electro-transfection reagent to resuspend cells.

Then adding Sug Cas9/gRNA2 expression vector plasmid and Sug linearized Donor plasmid (containing CDK 13 gene target sequence homologous arm sequence and GFP expression cassettes), and slowly adding electro- transfection system (including cells, transfection reagent and plasmid) into the electric rotating cup along the wall, so as to avoid reducing transfection efficiency due to air bubbles.

C.

After transfection, adding 500uL complete medium (20% FBS+DMEM) into the electric rotating cup while gently blowing the cells.

Then transferring them to a preheated 6cm petri dish with 5mL culture solution and cultivating them in an incubator containing 5% CO» at 37.5 ‘C.

After a 48-hour electro-transfection, the plate was plated when the confluence of cells was about 90%. The recommended density of plates was about 100 cells per 10cm dish, and the culture medium was changed every 3 days.

After plating, the cells were cultured for about 10 days, and the formation of clone sites with LU102620 appropriate size could be observed.

Finding clone sites under microscope, marking them with marker pen, and the miscellaneous cells beside clone sites were removed.

The culture medium was then dumped and washed twice with PBS.

Covering the marked clone sites with metal clone rings.

Two drops (200uL gun) of 0.1% trypsin digestion solution preheated at 37°C were added to each clone ring.

After digestion at 37°C for 3min, adding appropriate amount of complete medium to terminate the digestion reaction.

The digested cells were inoculated into a 24-well culture plate for culture.

After about 3 days of culture, when the convergence degree of cells in the 24-well plate reached about 90%, digesting the cells again.

Then some cells were inoculated into the 12-well plate for further cultivation, and other cells were added into cell lysis buffer (40mMTris-Cl, 0.9% Nonidet P-40, 0.9% Triton X-100, 0.4mg/mL protease K) to extract the cell genome for identification of positive cells.

The extracted genome was sent to a biological detection company for detection.

The results showed that the gRNA1 and gRNA2 designed in the present invention can successfully modify the CDK13 target sequence based on CRISPR-Cas9 technology, providing technical support for the construction of CDK13 gene-deficient mouse models, which is of great significance for the construction of CDK13 genetically modified mouse models.

The above embodiments only describe the preferred mode of the invention, but do not limit the scope of the invention.

On the premise of not departing from the design spirit of the invention, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the invention shall fall within the protection scope determined by the claims of the invention.

SEQUENCE LISTING LU102620 <110> The Second Affiliated Hospital of Wenzhou Medical University <120> A gRNA and Related Kit and Application <130> PT1042 <160> 4 <170> BiSSAP 1.3.6 <210> 1 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> gRNA that can recognize the CDK13 gene nucleotide sequence first <400> 1 agacagcucu uacuagacuu ugg 23 <210> 2 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> gRNA that can recognize the CDK13 gene nucleotide sequence second <400> 2 ccucagacua uaucuacuaa ggg 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence of the DNA molecule first <400> 3 agacagctct tactagactt tgg 23

<210> 4 LU102620 <211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> nucleotide sequence of the DNA molecule second

<400> 4 cctcagacta tatctactaa ggg 23

Claims (6)

THE CLAIMS: LU102620

1. A gRNA, characterized in that its nucleotide sequence comprises one or two sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2.

2. The biological materials related to the gRNA as stated in Claim 1, characterized by comprising (1) a DNA molecule encoding the gRNA of Claim 1; (2) an expression cassette including the DNA molecule in (1); (3) expression vectors, including DNA molecules in (1) or expression cassettes in (2).

3. The biological materials according to Claim 2, characterized in that the nucleotide sequence of the DNA molecule includes one or two sequences shown in SEQ ID NO: 3 and SEQ ID NO:

4.

4. The biological materials as stated in Claim 2, characterized in that the expression cassette further comprises DNA molecules that can encode Cas9.

5. A kit, characterized by comprising the gRNA stated in Claim 1 or biological materials based on any one of Claims 2-4.

6. The kit as stated in Claim 5, characterized by further comprising matched detection reagent, which is composed of the following components: template 1uL, reaction buffer 2.SuL, upstream primer/downstream primer 0.5uL, Mg** 2.5uL, dNTPs 0.5uL and Taq DNA polymerase

0.25uL; finally, ddH2O is supplemented to 20uL.