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A gene expression regulation method and regulation system based on type I-F CRISPR/Cas

A gene expression and target gene technology, applied in the field of molecular biology, to achieve high transcriptional activation efficiency and strong specificity

Active Publication Date: 2022-04-15
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there has not been any research or report on whether the Type I-F CRISPR system has a role in gene expression regulation and its corresponding application value. Whether it can become a new generation of gene regulation tool is still unknown.

Method used

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  • A gene expression regulation method and regulation system based on type I-F CRISPR/Cas
  • A gene expression regulation method and regulation system based on type I-F CRISPR/Cas
  • A gene expression regulation method and regulation system based on type I-F CRISPR/Cas

Examples

Experimental program
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Effect test

Embodiment 1

[0127] The sequence optimization of embodiment 1 PaeCascade

[0128] The PaeCascade sequence is derived from the type I-F CRISPR complex of Pseudomonas aeruginosa. Jennifer A. Doudna's team published an article entitled "RNA-guided complex from a bacterial immune system enhances target recognition throughseed sequence interactions" in "Proc Natl Acad Sci" in 2011, which reported that PaeCascade can bind dsDNA sequences in vitro. Its source strain is Pseudomonas aeruginosa.

[0129] Since the sequences of the four proteins (Cas8f1, Cas5f1, Cas7f1, and Cas6f) at the PaeCascade site are regulated by prokaryotic polycistrons, they are prokaryotic preferred codon coding sequences (SEQ ID NO.1). Therefore, in order to promote the expression of PaeCascade in mammalian cells, its sequence characteristics should be optimized. There are three principles for the transformation: 1) Split out individual proteins, each of which is optimized for mammalian codons; 2) Remove unexpected eukar...

Embodiment 2

[0134] Embodiment 2 constructs recombinant plasmid

[0135] 1. Construct the recombinant plasmid pxCMV-hCas5f1-PGK-hCas8f1 (sequence shown in SEQ ID NO.9)

[0136] The AscI and MluI restriction sites and the NotI and HindIII restriction sites were connected to the improved pxCMV plasmid respectively, hCas8f1 and hCas5f1 containing the NLS sequence (PKKKRKV) were connected and the pxCMV-hCas5f1-PGK-hCas8f1 plasmid (SEQ ID NO .9).

[0137] The improved pxCMV used was derived from the px601 plasmid, in which an AscI restriction site was added behind the CMV promoter, and the PGK promoter and NotI restriction site were constructed by inserting the original KpnI and HindIII sites.

[0138] 2. Construct the recombinant plasmid pxCMV-hCas6f-PGK-hCas7f1 (sequence shown in SEQ ID NO.10):

[0139] Linked to the improved pxCMV plasmid through AscI and MluI restriction sites and NotI and HindIII restriction sites respectively, hCas7f1 and hCas6f containing NLS sequence (PKKKRKV) were co...

Embodiment 3

[0161] Example 3 Construction of PaeCascade-VPR Transcription Activation System

[0162] The PaeCascade-VPR transcriptional activation system includes the following three components:

[0163] (1) fusion plasmid pxCMV-hCas5f1-PGK-hCas8f1 (sequence such as SEQ ID NO.9),

[0164] Or fusion plasmid pxCMV-hCas6f-PGK-hCas7f1 (sequence such as SEQ ID NO.10);

[0165] (2) fusion plasmid pxCMV-hCas7f1-VPR-PGK-hCas6f (sequence such as SEQ ID NO.11),

[0166] Or fusion plasmid pxCMV-hCas5f1-VPR-PGK-hCas8f1 (sequence such as SEQ ID NO.12),

[0167] Or fusion plasmid pxCMV-hCas8f1-VPR-PGK-hCas5f1 (sequence such as SEQ ID NO.13),

[0168] Or fusion plasmid pxCMV-hCas6f-VPR-PGK-hCas7f1 (sequence such as SEQ ID NO.14);

[0169] (3) The crRNA expression plasmid is pLenti-DR(hCas6f)-EV (SEQ ID NO.16).

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Abstract

The invention discloses a gene expression regulation method and a gene expression regulation system based on Type I‑F CRISPR / Cas. The present invention shows for the first time that the Cascade complex belonging to the Type I-F CRISPR / Cas system, such as the PaeCascade complex, can efficiently bind to the target site in mammalian cells; at the same time, the present invention conducts the Type I-F CRISPR / Cas system The mammalian expression system has been optimized so that it can efficiently recruit transcriptional activators or transcriptional repressors to target sites in mammalian cells, and successfully constructed a transcriptional activation system and a transcriptional repression system. The invention makes it possible to apply the Type I-F CRISPR / Cas system in the gene regulation of mammalian cells, and provides a necessary tool for gene transcription regulation based on the Type I-F CRISPR / Cas system.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to a type I-F CRISPRCas system-based gene expression regulation method, gene expression regulation system and application. Background technique [0002] Gene expression regulation and gene editing are key technologies in the field of targeted gene therapy. Gene expression regulation is different from gene editing. Gene expression regulation refers to the regulation and control of gene expression in organisms, so that the process of gene expression in cells is in an orderly state in time and space, and a complex process that responds to changes in environmental conditions. The regulation of gene expression can be carried out at multiple levels, including gene level, transcription level, post-transcription level, translation level and post-translation level regulation. The CRISPR / Cas system is a reported high-efficiency gene-level manipulation method, which...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N9/22C12N15/85C12N15/65C12N15/90
CPCC12N15/113C12N9/22C12N15/85C12N15/65C12N15/907C12N2310/20
Inventor 松阳洲陈昱僖刘嘉琪梁普平
Owner SUN YAT SEN UNIV
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