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Novel genome editing tool (Lb2Cas12a-RVR) and construction method and application method thereof

A genome editing and gene editing technology, applied in the field of new genome editing tools and its construction, can solve the problems of low activity and low fidelity of specific gene editing

Inactive Publication Date: 2020-09-11
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies existing in the prior art, the object of the present invention is to provide a novel modified genome editing tool and its construction method and application method. The problem of low activity and low fidelity of point-specific gene editing is helpful for in-depth research on the screening of clinical tumor treatment targets, drug development, and correction of pathogenic mutations

Method used

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  • Novel genome editing tool (Lb2Cas12a-RVR) and construction method and application method thereof
  • Novel genome editing tool (Lb2Cas12a-RVR) and construction method and application method thereof
  • Novel genome editing tool (Lb2Cas12a-RVR) and construction method and application method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Construction of pcDNA3.1-hLb2Cas12a-RVR plasmid

[0039] (1) Using the PCR method, set the corresponding mutation N512R+K518V+N522R on the primer, amplify the sequence containing the RVR mutation site on the pY011 (pcDNA3.1-hLb2Cas12a) vector, and pass the amplified fragment and backbone through ClonExpressII One Step Cloning Kit gets the vector pcDNA3.1-hLb2Cas12a-RVR after mutation.

[0040] (2) The PCR system used in the plasmid construction experiment is as follows:

[0041] Template: 10ng; Forward Primer: (10μM) 1μl; Reverse Primer: (10μM) 1μl;

[0042] dNTP: 0.5μl; DNA polymerase (Vazyme, P501): 0.5μl; 5x Buffer: 10μl; RNase-Water fill up to 50μl.

[0043] The PCR program is as follows:

[0044] 95℃, 3min; 95℃, 15sec, 60℃, 15sec, 72℃, 1min; 35cycles; 72℃, 3min.

[0045] The application method of a Lb2Cas12a-RVR gene editing tool of this embodiment:

[0046] Step 33: Including the construction of crRNA directed against the DNA target of a certain disease-causing gen...

Embodiment 2

[0054] Example 2: Establishment of a GFP reporter system for pcDNA3.1-hLb2Cas12a-RVR activity test

[0055] (1) Construct a pSin-GFP lentiviral vector, which contains the GFP gene (as shown in SEQ ID NO. 4), IRES and Puromycin resistance genes. pSin-GFP and the helper plasmid were co-transfected into HEK-293T cells, the virus supernatant was collected, filtered with a 0.45μm filter, and aliquoted and stored at -80°C for later use.

[0056] (2) Recovery of HEK-293 cells: Take out the tube of frozen HEK-293 cells from liquid nitrogen, immediately put it in a 37°C water bath, shake it slightly, wait until the liquid is completely melted (about 1~1.5min), take it out Wipe and disinfect with 75% alcohol and place it on the ultra-clean workbench; transfer the above cell suspension to a 15ml centrifuge tube containing 5ml culture medium, centrifuge at 1500rpm for 5min; discard the supernatant, add 1ml culture medium to resuspend the cells . Transfer to a 10cm petri dish containing 10ml ...

Embodiment 3

[0062] Example 3: Determination of the efficiency of pcDNA3.1-hLb2Cas12a-RVR at a specific site

[0063] (1) pJET-U6-sgRNA design: design crRNA at different positions of the GFP gene sequence according to the requirements of subsequent evaluation;

[0064] (2) HEK-293-SC1 cells were counted and then seeded in a 12-well plate with a cell density of 1.8×105 cells / well;

[0065] (3) After 24h of cell growth, the transfection reagent TurboFect (Thermo Fisher, #R0531) was used to transfect pcDNA3.1-hLb2Cas12a-RVR (750ng) and pJET-U6-crRNA (250ng);

[0066] (4) Eukaryotic cell transfection method: After mixing the plasmids to be transfected in proportion, mix in 50μl DMEM containing 1.5μl of transfection reagent Turbofect, pipetting and mixing, and after 15 minutes at room temperature, add 1ml DMEM containing (Gibco, C11995500CP) + 10% FBS (Gibco, 16000 044) medium, and 1.8×105 cells / well of HEK-293-SC1 cells were transfected in a 12-well plate.

[0067] (5) Change the fresh medium 24h after...

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Abstract

The invention discloses a novel genome editing tool (Lb2Cas12a-RVR) and a construction method and application method thereof. The novel genome editing tool (Lb2Cas12a-RVR) is characterized by comprising coding plasmids pcDNA3.1-hLb2Cas12a-RVR for coding and expressing an Lb2Cas12a-RVR mutein, wherein the DNA sequence of a coding protein is shown as SEQ ID NO.1. The novel reformed genome editing tool and the construction method and application method thereof aim to solve the problems of low activity and low fidelity of specific gene editing at precise sites, contribute to deep research on screening clinical oncotherapy targets, developing medicines, correcting pathogenic mutation and the like, have genome editing activity in human cells and have better PAM flexibility, high fidelity and special cutting manners.

Description

Technical field [0001] The invention relates to a new type of genome editing tool (Lb2Cas12a-RVR) and its construction method and application method. Background technique [0002] As a new genome editing tool, Cas12a, a new member of the CRISPR family, has received extensive attention in recent years and has emerged in the field of gene therapy in human cells and animal and plant models. Cas12a-mediated targeted genome engineering technology has achieved a wide range of research and medical applications. However, existing researches mostly focus on FnCas12a, AsCas12a and LbCas12a. Due to this limitation, mammalian-related genome editing research also focuses on AsCas12a and LbCas12a. However, for the extensive use of precise site-specific human genome editing Application, limited target selection spectrum, low activity and low fidelity will still be the bottleneck of its mediating application. Therefore, there is an urgent need to develop new Cas12a nucleases with improved char...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/66
CPCC12N15/79C12N2310/20
Inventor 谷峰刘潇宇林莉
Owner WENZHOU MEDICAL UNIV
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