Efficient gene knockout vector and application thereof

A gene knockout and vector technology, applied in the field of gene editing, can solve problems such as unclear function and unclear copy number, and achieve the effect of reducing damage

Active Publication Date: 2020-05-19
浙江吉诺赛百尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its function in mammals remains unclear, and in domestic pigs, even the copy number remains unclear

Method used

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  • Efficient gene knockout vector and application thereof
  • Efficient gene knockout vector and application thereof
  • Efficient gene knockout vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Construction of gene knockout vector

[0032]U6 promoter-driven gRNA cloning vector pX330 was purchased from Addgene (Plasmid No. 42230; Watertown, MA, USA). gRNAs targeting exon 1 of porcine pLeg1a (XM_003121211.1 / XP_003121259.1) and pLeg1b (XM_021074892.1 / XP_020930551.1) were designed using the online gRNA design tool (http: / / crispr.mit.edu).

[0033] Firstly, the gRNA recombination vectors of pX330-pLeg1a and pX330-pLeg1b were respectively constructed using the BpiI restriction site in pX330. The primers used for gRNA-pLeg1a are forward (SEQ ID NO:5): 5'-CACCGCATGGCTGGCAATATACTAC-3', reverse (SEQ ID NO:6): 5'-AAACGTAGTATATTGCCAGCCATGC-3'; and for gRNA-pLeg1b The primers were forward (SEQ ID NO:7): 5'-CACCGCTGGCATTACCTTGAGAGAC-3', reverse (SEQ ID NO:8): 5'-AAACGTCTCTCAAGGTAATGCCAGC-3'.

[0034] A primer containing U6- The 380 base pair fragment of gRNA-pLeg1b was then inserted into the XbaI site of the pX330-pLeg1a vector to generate a recombinant vec...

Embodiment 2

[0035] Example 2: Culture, transfection and screening of pig fetal fibroblasts

[0036] Porcine fetal fibroblasts (PFFs) were isolated from 32-day-old male Chinese experimental minipig embryos. These primary cells were cultured in high glucose Dulbecco's Medium Eagle medium (Gibco, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS; Gibco).

[0037] All animal experiments were performed according to the guidelines established by the China Animal Protection and Protocol Committee and were approved by the Experimental Animal Welfare Committee of Zhejiang University (Zhejiang Province, China). Transfection of PFF with pX330-pLeg1a-pLeg1b was performed using a Lonza Nucleofector. The day before transfection, PFFs were thawed and cultured. Then use 3 μg pMax (Lonza, Walkersville, MD, USA) to carry out transfection under different parameters (CA-137, CL-133 and DO-113), transfect about 1×10 6 PFF cells. The transfection effect was evaluated by the fluorescence intensi...

Embodiment 3

[0039] Example 3: Detection of Gene Modification and Integration of Exogenous Genes

[0040] Genomic DNA was extracted from each cell clone using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA), and then detected by PCR using high-fidelity Phusion polymerase (Thermo Fisher Scientific), and the pLeg1a-specific primers used were : 5'-CCCCCGCTGTGTGGAATAAGAG-3'(SEQ ID NO:11) and 5'-GCCCCACTGCAGTTTTTAGTAGC-3'(SEQ ID NO:12), the pLeg1b-specific primers used are: 5'-ACAAGGGAAGTGCCCTATTACC-3'(SEQ ID NO : 13) and 5'-TGATGTACAGGCAATCCCCA-3' (SEQ ID NO: 14).

[0041] PCR products were gel purified and then subjected to Sanger sequencing (BGI Genomics, Shenzhen, China).

[0042] In addition, the genomic DNA of the screened double gene knockout cell clones was also tested by PCR to verify the integration of the vector plasmid. The primers used were: Cas9-F (SEQ ID NO: 15): 5'-CATCGAGCAGATCAGCGAGT-3' and Cas9-R (SEQ ID NO: 16): 5'-CGATCCGTGTCTCGTACAGG...

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Abstract

The invention provides an efficient gene knockout vector based on a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/Cas9) system. The efficient gene knockout vector comprises atleast two DNA (deoxyribonucleic acid) fragments which are serially connected, target at different genes or different loci of genes and are designed according to sgRNA (small guide ribonucleic acid) target loci. Simultaneous knockout of double genes or multiple genes can be efficiently, rapidly and conveniently achieved, and meanwhile, damage to transfection cells can be reduced. The invention further provides a method for knocking out a pig liver enriched gene 1, and pig fibroblast of which diallele genes pLeg1a and pLeg1b are simultaneously knocked out.

Description

technical field [0001] The present invention relates to the field of gene editing, in particular to a method for simultaneously knocking out multiple genes based on CRISPR / Cas9 technology and a method for preparing pig fibroblasts knocked out of pig liver enrichment gene 1. Background technique [0002] Based on the genome editing technology mediated by CRISPR (Clustered regularly interspaced short palindromicrepeats) / Cas9 system, it is following zinc-finger nucleases (Zinc-finger nucleases, ZFNs) and transcription activator-like effector nucleases (Transcription activator-like effector nucleases, TALEN ) The third-generation genome editing technology after ) is based on the transformation of the acquired immune system of bacteria. CRISPR / Cas9 is a gene editing technology derived from the acquired immune system mediated by bacteria or archaea regularly clustered interspaced short palindromic repeats CRISPR (clustered regularly interspaced short palindromic repeats). This te...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/113C12N15/90C12N5/10A01K67/027
CPCA01K67/0276A01K2207/15A01K2217/075A01K2227/108A01K2267/03C07K14/47C12N15/113C12N15/8509C12N15/907C12N2310/20
Inventor 贺津彭金荣
Owner 浙江吉诺赛百尔生物科技有限公司
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