Method for carrying out gene editing and expression regulation by utilizing Cas splitting system

A technology for gene editing and encoding genes, which can be used in chemical instruments and methods, biochemical equipment and methods, genetic engineering, etc., and can solve the problems of low efficiency of Cas9 gene editing.

Active Publication Date: 2016-10-12
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Cas9 gene editing efficiency obtained by these two split systems is still relatively low, and further improvement is needed to improve the activity of Cas9 after recombination

Method used

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  • Method for carrying out gene editing and expression regulation by utilizing Cas splitting system
  • Method for carrying out gene editing and expression regulation by utilizing Cas splitting system
  • Method for carrying out gene editing and expression regulation by utilizing Cas splitting system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Example 1. Obtaining and functional verification of split Cas9 protein group and recombinant vector group

[0112] Utilizing the detachable feature of the Cas9 protein functional domain, the coding region of a Cas9 gene is split into two fragments (Cas9N and Cas9C) at the N-terminus and C-terminus, which are respectively combined with the inteinN and InteinC in the cell. The Intein domain catalyzes protein cleavage, allowing it to recombine into a complete Cas9 protein in the cell, followed by gene editing and gene expression regulation.

[0113] figure 1 It is a schematic diagram of Cas9 protein splitting, and it is different amino acid sequences obtained by splitting the amino acid sequence of Cas9 protein from at least one of the following positions; the positions are between 203-204, 468-469, and 713-714 Between bits and between 1153-1154th.

[0114] The Cas9 protein used in the embodiments of the present invention is from Streptococcus pyogenes, namely hpCas9, it...

Embodiment 2

[0170] Example 2. Obtaining and functional verification of split dCas9 protein group and recombinant vector group

[0171] The split Cas9 proteins used in the following experiments are all dCas9 proteins, which cannot be used for gene editing, and can only guide VP64 (the amino acid sequence is sequence 9) or KRAB (the amino acid sequence is sequence 10) to regulate gene expression. In addition, the splitting site of the following experimental group is the position between amino acids 713-714 of the dCas9 protein.

[0172] The amino acid sequence of the dCas9 protein is sequence 6 in the sequence listing, and the nucleotide sequence of its coding gene dCas9 is sequence 5 in the sequence listing.

[0173] The amino acid sequence of dCas9N is the 1-713th position of Sequence 6 in the Sequence Listing, and the nucleotide sequence of its coding gene dCas9N is the 1st-2139th position of Sequence 5 in the Sequence Listing;

[0174]The amino acid sequence of dCas9C is No. 714-1368 o...

Embodiment 3

[0210] Example 3. Comparison of Genome Editing without Intein-mediated Split Cas9 System and Intein-mediated Split Cas System

[0211] In view of the article that the split Cas9 protein can be automatically recombined in the cell, this implementation test splits and tests the 714th and 1154th amino acid positions of SpCas9 selected in the present invention, and compares it with Example 1.

[0212] 1. The 714-site split of Spcas9 uses Intein to mediate the efficiency comparison

[0213] 1. Construction of recombinant vector

[0214] The pCAG-SpCas9N714 plasmid has a nucleotide sequence of sequence 39, wherein nucleotides 4635-6773 of sequence 39 are the gene encoding protein SpCas9N714, the vector expresses protein SpCas9N714, and the amino acid sequence of the protein is sequence 2 1-713 .

[0215] The pCAG-SpCas9C714 plasmid has a nucleotide sequence of sequence 40, wherein the 4638-6599 nucleotides of the sequence 40 are the gene encoding the protein SpCas9C714, the vector...

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Abstract

The invention discloses a method for carrying out gene editing and expression regulation by utilizing a Cas splitting system. A protein group is provided for splitting a Cas9 protein amino acid sequence from different sites to form two-section proteins which form a protein group. Proved by an experiment, an Intein-mediated Ca9 splitting system can efficiently realize gene editing and transcriptional activation regulation on a gene circuit. According to the method disclosed by the invention, the CaS9 splitting system is combined with a tumor cell specific promoter, so that specific detection can be carried out on bladder cancer cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for gene editing and expression regulation using a split Cas system. Background technique [0002] Gene editing and regulation of gene expression are key technologies in the field of targeted gene therapy. The RNA-guided CRISPR / Cas9 system has become a new tool for genome editing since 2013, and because its vector construction process is more convenient than the other two editing tools TALE and zinc finger protein, it has been sought after by researchers in this field . CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is a newly emerging technology that uses RNA-guided Cas nucleases to perform specific DNA modifications on targeted genes. The CRISPR / Cas system is widely distributed in the genomes of bacteria and archaea, and is an adaptive immune system formed during evolution, which can degrade invading virus or plasmid DNA. In this system, crRNA (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C07K19/00C12N15/85
CPCC12N9/22C12N15/85C07K2319/00C07K2319/92C12N2800/107C12N2810/10C12N15/8216C12N15/8213C12N15/90C12N2710/10343C12N7/00A61K48/0075A61K48/005C12N15/86
Inventor 谢震马大程彭曙光
Owner TSINGHUA UNIV
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