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Novel method for gene assembly using improved CRISPR-Cpf1

A new method, gene technology, applied in the field of gene assembly using improved CRISPR-Cpf1, can solve the problems of low probability of recombination, reduced fragment connection efficiency, high cost, etc., to save synthesis cost, improve recombination efficiency and repeatability , the effect of improving the efficiency of recombination

Active Publication Date: 2019-05-31
HUBEI UNIV
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Problems solved by technology

[0003]In 2016, Zhao Guoping’s research group at the Institute of Plant Physiology and Ecology, Chinese Academy of Sciences developed a C-Brick gene assembly method based on nucleic acid editing enzyme FnCpf1 and biological bricks, using nucleic acid editing enzymes FnCpf1 replaces the traditional restriction endonuclease to cut nucleic acid fragments, but the nucleic acid editing enzyme FnCpf1 is inaccurate when cutting, and will produce uneven sticky ends of 5-8 bases, thereby reducing the ligation efficiency of fragments, so In the C-Brick method, the gene assembly efficiency can only reach about 20%. The recombination efficiency of this assembly method is low, and the probability of obtaining the correct recombinant is small. Moreover, the sgRNA used in the CRISPR-Cpf1 method of the prior art is a seed sequence of 24nt sgRNA is more expensive and more complicated to manipulate

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Examples

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Embodiment 1

[0049] Example 1 Construction of expression vector for nucleic acid editing enzyme FnCpf1

[0050] According to the T5 exonuclease-mediated cloning method, PCR amplification was performed on the nucleic acid programming enzyme FnCpf1 gene fragment and the expression vector pET23a. After the product was detected and recovered by the agarose gel, the nucleic acid concentration was measured with a Nanodrop 8000 spectrophotometer. The fragments and the vector fragments were mixed in a molar ratio of 3:1, T5 exonuclease was added and reacted in an ice-water mixture for 5 minutes, and then the conventional transformation of E. coli was carried out. Pick a single colony separately into 3ml of lysic broth liquid medium supplemented with ampicillin, culture for several hours at 37°C, extract plasmid DNA according to the alkaline lysis method, and then perform agarose gel electrophoresis detection, the size is correct and PCR identified contains The plasmid of the target fragment is sent t...

Embodiment 2

[0052] Example 2 Expression and identification of FnCpf1 nucleic acid editing enzyme in E. coli

[0053] Transform the correctly sequenced recombinant plasmid into Escherichia coli BL21(DE3) expression strain, pick a single colony to inoculate 100ml of lysic broth medium supplemented with ampicillin, and cultivate to OD at 37°C 600 For 0.6-0.8, add 1M IPTG (isopropyl-β-D-thiogalactoside) at a volume ratio of 1%, culture overnight at 18°C, 200rpm shaking; collect the bacteria and resuspend the bacteria in lysis buffer, After bacteriostasis by ultrasound, centrifuge, add 5× protein loading buffer to the centrifuged supernatant and pellet respectively, treat in a metal bath at 100°C for 10 minutes, and then perform SDS-PAGE sodium dodecyl sulfonate-polyacrylamide gel Electrophoresis, the results are as image 3 .

Embodiment 3

[0054] Example 3 Purification of nucleic acid editing enzyme FnCpf1 with nickel beads

[0055] Take a single colony containing the recombinant plasmid and inoculate it into 200ml lysing broth medium supplemented with ampicillin, and cultivate to OD at 37°C 600 Add 1% IPTG (isopropyl-β-D-thiogalactoside) when it reaches 0.6-0.8, and induce overnight at 18°C; collect E. coli cells, resuspend the cells in lysis buffer, and then ultrasonically break Cells, the crude lysate and the affinity resin are thoroughly mixed to bind the target protein to the nickel beads, and then the recombinant protein is eluted with different concentrations of imidazole buffer, and the effluent obtained by gradient elution is subjected to sodium dodecyl sulfonate- Polyacrylamide gel electrophoresis, the results are as Figure 4 .

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Abstract

The invention discloses a novel method for gene assembly using improved CRISPR-Cpf1. The method includes utilizing a DNA gene editing enzyme FnCpf1 from a Francisella tularemia noticed U112 strain (gene sequence number: MF193599) for gene recombinant cloning. The seed sequence of crRNA (an RNA sequence generated by transcription of regular clustering spaced CRISPR) from 23 nucleotides reported inthe literature to 18 without affecting the FnCpf1 cleavage efficiency. At the same time, oligo(dN)6 (oligonucleotide hexamer) is used as the viscous end of the cleavage target site, the problem of terminal heterogeneity after FnCpf1 digestion is solved, the assembly efficiency of exogenous DNA fragments is remarkably improved, and the DNA fragment assembly efficiency reaches about 85%. The gene editing enzyme FnCpf1 can be replaced with other nucleic acid editing enzymes Cpf1 such as AsCpf1 and LbCpf1.

Description

Technical field [0001] The invention belongs to the field of molecular biology or enzymology, and is particularly a new method for gene assembly using improved CRISPR-Cpf1. Background technique [0002] The CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and associated protein complexes) is an adaptive immune system widely present in bacteria and archaea. In 1987, scientists first discovered the CRISPR sequence. In 2010, researchers began to gradually elucidate the biological function of this sequence and apply it to gene editing. Based on this system, researchers have discovered a variety of nucleic acid editing enzymes and developed a large number of in vivo and in vitro gene editing technologies. In 2013, Zhang Feng et al. first reported the application of the CRISPR-Cas9 system in mammalian genome editing. In September 2015, the research team led by Zhang Feng found another nucleic acid editing enzyme, Cpf1. This nucleic acid editing enzym...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/90
CPCY02A50/30
Inventor 马立新董梦洁翟超韩瑞王亚平
Owner HUBEI UNIV
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