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A responsive DNA editing system based on CRISPR/cpf1 and its application

An editing system, cpf1 technology, applied in the field of responsive DNA editing systems, can solve problems such as off-target and precise activity regulation, and achieve the effect of reducing the off-target rate

Active Publication Date: 2022-08-09
TIANJIN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After the CRISPR / Cpf1 system enters the cell, it will have a persistent editing effect on the genome. The current gene editing technology cannot precisely regulate its activity, which may cause serious off-target phenomena

Method used

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  • A responsive DNA editing system based on CRISPR/cpf1 and its application
  • A responsive DNA editing system based on CRISPR/cpf1 and its application
  • A responsive DNA editing system based on CRISPR/cpf1 and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1.1 Construction of the Optic-split-AsCpf1 system

[0046] Optic-split-AsCpf1 consists of a three-plasmid system such as figure 1 As shown, firstly by analyzing the crystal structure of AsCpf1-crRNA-target DNA (PDB: 5B43), the division site that controls the activity of AsCpf1 was determined, and AsCpf1 was divided into two parts: N-terminal and C-terminal, which were connected to each other under blue light stimulation and could interact with each other. On the CIBN and CRY2PHR, the SV40NLS-Flag-AsCpf1 N-terminal-CRY2PHR and CIBN-HA-AsCpf1 C-terminal-SV40NLS sequences were constructed by the method of total gene synthesis, and cloned into pCDNA3.1 using seamless cloning technology + downstream of the vector CMV promoter. The AsCpf1 crRNA was cloned downstream of the human U6 promoter and expressed by U6.

[0047] In the Optic-split-AsCpf1 system, the two parts are dissociated separately under dark conditions to inhibit the function of AsCpf1, and the interaction occu...

Embodiment 2

[0069] 2.1 Construction of Optic-CRIPSR-Activin system

[0070] Optic-CRISPR-Activin consists of a three-plasmid system such as Figure 8 As shown, the mutations of AsCpf1 D908A and E993E make it lose its endonuclease activity but retain crRNA processing activity, so it is called dead AsCpf1 (dAsCpf1). The CIBN-SV40NLS-FLAG-dAsCpf1-CIBN-NLS fusion protein was constructed as a targeting element, and on the other hand, CRY2PHR was fused with the transcription activator sp65×3-HSF1 as an effector element. The AsCpf1 crRNA was cloned downstream of the human U6 promoter and expressed by U6.

[0071] Under dark conditions, crRNA guides dAsCpf1 protein to the designated position in the genome, but the activation element is free in the nucleus; under blue light irradiation, CIBN recruits CRY2PHR and carries the activation element to the designated position to activate gene transcription.

[0072] 2.2 Construction of Optic-CRISPR-Rep system

[0073] Optic-CRISPR-Rep is similar in co...

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Abstract

The present invention relates to a CRISPR / Cpf1-based responsive DNA editing system and its application, comprising a functional protein A and a stimulus-responsive protein C connecting element, a functional protein B and a stimulus-responsive protein D connecting element and a guide element; wherein, the stimulus-responsive protein C and stimuli-responsive protein D can bind to each other under stimulation, and the binding is released after the stimulation disappears; functional protein A and functional protein B linked to stimuli-responsive proteins C and D are proteins to be connected, and functional protein A and functional protein B are among the proteins to be linked. One or both of them are Cpf1. The beneficial effects of the present invention are that optogenetics and DNA editing are combined to realize precise time and space control of DNA editing. By establishing a CRISPR / Cpf1-based responsive gene editing system and method, gene editing and transcriptional regulation can be achieved. Conditional on and off, thereby reducing the off-target rate, which is of great significance and research value in the field of gene editing.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a CRISPR / Cpf1-based responsive DNA editing system and its application. Background technique [0002] Deoxyribonucleic acid (DNA) is an important carrier of genetic information, carrying the genetic information necessary for the synthesis of RNA and protein, and is an essential biological macromolecule for the development and normal operation of organisms. Gene editing technology is an emerging genetic engineering technology that can precisely modify the DNA of organisms. The third-generation gene editing technology represented by CRISPR / Cas9 can knock out, knock in, and single-base gene loci. Replacement and other operations, with low cost, are becoming mainstream technologies in basic research and clinical applications. At present, a large number of studies have further expanded and optimized the CRISPR system, and then constructed a variety of gene editing tools that can be applie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/90
CPCC12N15/85C12N15/902C12N2800/107
Inventor 马信龙赵杰
Owner TIANJIN HOSPITAL
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