A kind of crispr/cas gene editing system and its preparation method and application

A gene editing, pcdna3.1-ljcas9 technology, applied in the field of gene editing, can solve problems such as further improvement of efficiency

Active Publication Date: 2021-05-11
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Cong et al. (MultiplexGenome Engineering Using CRISPR / Cas Systems.Science.2013) and Mali et al. (RNA-guided human genome engineering via Cas9.Science.2013) proved that the Cas9 system can be effective in 293T, K562, iPS and other cells. Targeted enzyme digestion, the efficiency of non-homologous recombination (NHEJ) and homologous recombination (HR) is between 3-25%, which is equivalent to TALEN enzyme digestion, but the efficiency still needs to be further improved

Method used

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  • A kind of crispr/cas gene editing system and its preparation method and application
  • A kind of crispr/cas gene editing system and its preparation method and application
  • A kind of crispr/cas gene editing system and its preparation method and application

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preparation example Construction

[0031] The invention provides a method for preparing the gene editing system, comprising the following steps:

[0032] Insert the DNA fragment encoding LjCas9 into the initial plasmid pcDNA3.1(+) to construct the pcDNA3.1-LjCas9 plasmid;

[0033] The sgRNA general expression frame DNA fragment was inserted into the initial plasmid pUC57 to obtain the pLjCas9-sgRNA plasmid.

[0034] In the present invention, the insertion site of the DNA fragment encoding LjCas9 is between the BamH I restriction site and the EcoR I restriction site of the initial plasmid pcDNA3.1 (+); in the present invention, the insertion Preferably, the DNA fragment encoding LjCas9 and the initial plasmid pcDNA3.1(+) are respectively subjected to double digestion and then ligated; the enzymes used for the double restriction are BamH I enzyme and EcoR I enzyme. In the present invention, the restriction enzyme digestion system preferably includes 1 μL of BamH I enzyme, 1 μL of EcoRI enzyme, 1 μg of DNA fragme...

Embodiment 1

[0043] Construction of CRISPR / Cas gene editing system

[0044] 1. Construction of plasmid vector pcDNA3.1-LjCas9

[0045] A 4185bp DNA fragment encoding LjCas9 (nucleotide sequence as shown in SEQ ID NO: 1) was synthesized and inserted into pcDNA3.1(+) by BamHI and EcoR I double digestion to obtain the pcDNA3.1-LjCas9 vector.

[0046] BamH I enzyme was purchased from Bao Biological Engineering (Dalian) Co., Ltd., catalog number 1010S, and EcoR I enzyme was purchased from Bao Bioengineering (Dalian) Co., Ltd., catalog number 1040S

[0047] Enzyme digestion system: 50 μL, reagents purchased from Treasure Bioengineering (Dalian) Co., Ltd.): BamH I enzyme 1 μL, EcoR I enzyme 1 μL, DNA fragment encoding LjCas9 or cDNA3.1(+) plasmid 1 μg, Buffer H 5 μL, add Double distilled water to 50 μL. The digestion temperature is 37°C, and the digestion time is 3h.

[0048] Connection steps and parameters:

[0049] Ligation system (10 μL, ligation reagent purchased from NEB Company, Cat. No...

Embodiment 2

[0082] Application of CRISPR / Cas gene editing system in gene editing of mammalian cell lines

[0083] Diqing sheep skin epithelial cell line DQSHS1 was purchased from the Kunming Cell Bank of the Chinese Academy of Sciences, number: KCB 94026.

[0084] 1. sgRNA target design

[0085] The coding region of the first exon of the sheep DKK2 gene (18259978-18260199, as shown below) was extracted from the sequence of sheep chromosome 6 (NCBI GI: 417531944), and the target site was designed.

[0086]

[0087]

[0088] Target sequence T1:

[0089] gctcacagttcggcagctcg(74-93) (SEQ ID NO: 12)

[0090] 2. Construction of sgRNA expression plasmid pair

[0091] First, send the company to synthesize single-stranded oligonucleotides according to the designed target sequence. The specific sequence is as follows:

[0092] Tj1-F: caccgctcacagttcggcagctcg (SEQ ID NO: 13)

[0093] Tj1-R: aaaccgagctgccgaactgtgagc (SEQ ID NO: 14)

[0094]Tj1-F and Tj1-R were annealed to obtain short dou...

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Abstract

The invention provides a CRISPR / Cas gene editing system and its preparation method and application, belonging to the technical field of gene editing. The CRISPR / Cas gene editing system includes a pcDNA3.1-LjCas9 plasmid and a pLjCas9-sgRNA plasmid. The application includes the following steps: determine the target sequence, design single-stranded oligonucleotide pairs; anneal to obtain double-stranded DNA fragments; connect to pLjCas9-sgRNA plasmid to obtain target sequence sgRNA expression vector; target sequence sgRNA expression vector and pcDNA3.1 ‑LjCas9 plasmid co-transfected cells and cultured. The gene editing system can specifically cut target DNA, and the editing efficiency is high.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a CRISPR / Cas gene editing system and its preparation method and application. Background technique [0002] ZFN, TALEN and CRISPR / Cas9 targeting technologies are several genome modification technologies that are relatively mature in research. (CRISPR) / CRISPR-associated (Cas) is a continuously evolving immune defense mechanism in bacteria and archaea. CRISPR / Cas9 uses a small RNA to recognize and cut DNA to degrade foreign nucleic acid molecules. Cong et al. (MultiplexGenome Engineering Using CRISPR / Cas Systems.Science.2013) and Mali et al. (RNA-guided human genome engineering via Cas9.Science.2013) proved that the Cas9 system can be effective in 293T, K562, iPS and other cells. Targeted enzyme digestion, non-homologous recombination (NHEJ), homologous recombination (HR) efficiency is between 3-25%, which is equivalent to TALEN enzyme digestion, but the efficienc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N15/85
CPCC12N15/85C12N15/907C12N2800/107C12N2810/10
Inventor 李和刚秦怀远赵金山辛京京张宁郝小静
Owner QINGDAO AGRI UNIV
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