Chromatin imaging method and chromatin imaging system based on Type I-F CRISPR/Cas

An imaging system, chromatin technology, applied in the field of molecular biology

Active Publication Date: 2020-10-30
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, there has not been any research or report on whether the Type I-F CRISPR system is suitable for chromatin imag

Method used

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  • Chromatin imaging method and chromatin imaging system based on Type I-F CRISPR/Cas

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Experimental program
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Effect test

Embodiment 1

[0100] The sequence optimization of embodiment 1 PaeCascade

[0101] 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.

[0102] 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

[0105] Embodiment 2 constructs recombinant plasmid

[0106] 1. Construct the recombinant plasmid pxCMV-hCas5f1-PGK-hCas8f1 (sequence shown in SEQ ID NO.7)

[0107] 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 .7).

[0108] 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.

[0109] 2. Construct the recombinant plasmid pxCMV-hCas6f-PGK-hCas7f1 (sequence shown in SEQ ID NO.8):

[0110] 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 con...

Embodiment 3

[0121] Example 3 Construction of PaeCascade-mScarlett Chromatin Imaging System

[0122] The PaeCascade-KRAB transcriptional repression system includes:

[0123] (1) pxCMV-hCas5f1-PGK-hCas8f1 plasmid (SEQ ID NO.7),

[0124] (2) pxCMV-hCas7f1-mScarlett-PGK-hCas6f plasmid (SEQ ID NO.9),

[0125](3) The crRNA expression plasmid is pLenti-DR(hCas6f)-EV (SEQ ID NO.10).

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Abstract

The invention discloses a chromatin imaging system based on a Type I-F CRISPR/Cas system. According to the present invention, the Cascade compound, such as the PaeCascade compound, belonging to the Type I-F CRISPR/Cas system is firstly researched and displayed, and can be effectively used for chromatin imaging; meanwhile, a Type I-F CRISPR/Cas system is subjected to mammal expression system optimization, the system is enabled to have efficient double-stranded DNA binding activity in mammalian cells, and fluorescent molecule is subjected to fusion expression to optimized Cascade subunit so as to successfully construct chromatin imaging system; the application of the Type I-F CRISPR/Cas system in chromatin imaging of mammalian cells becomes possible, and a necessary tool is provided for chromatin imaging 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-based chromatin imaging method, chromatin imaging system and application. Background technique [0002] In eukaryotic cells, genomic DNA is stored in the three-dimensional space of the nucleus in the advanced form of chromatin, so it is called "three-dimensional genome". Abnormalities in the three-dimensional genome structure are closely related to the occurrence of various human diseases. The three-dimensional structure of the genome is a dynamic structure that exhibits single-cell heterogeneity. Therefore, in the state of living cells, it is particularly important to use single-cell resolution to study the dynamics and functions of the three-dimensional genome structure. There are many important scientific issues waiting to be explored in the field of 3D genome. For example, what molecule determines the location of a particular...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/85C12N15/65C12N15/90G01N21/64
CPCC12N15/113C12N9/22C12N15/85C12N15/65C12N15/907G01N21/6428C12N2310/20
Inventor 松阳洲陈昱僖刘嘉琪梁普平
Owner SUN YAT SEN UNIV
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