Gene editing is a genetic engineering technique, which is pointed to a sequence without knowing the function of it, by altering the genetic gene of a living organism to reduce the effect of a particular gene function. By studying the effects on the organism, we can find the biological function of genes.
1. Gene knockout
Generally, it performs DNA level editing and used to build a knockout mouse model.
CRISPR / Cas9: as an acquired immune system for bacteria and archaebacteria, CRISPR / Cas9 system combats invasive viruses and plasmids through RNA mediated specific foreign genetic material. The use of Cas9 incision enzyme and a pair of sgRNA make DNA double strand in two similar incisions break, inducting cell non-homologous terminal attachment repair, and resulting in the target gene mutation.
CRISPR/Cas9 has been used as an effective tool for research, medical and other fields since its inception, replacing the "zinc finger endonuclease (ZFN)" and "transcriptional activator-like effector nuclease (TALEN)". SgRNA and cas9 mRNA are injected into the prokaryotic fertilized eggs and transplanted into the fake pregnant mice to obtain gene-edited mice for gene function studies.
Construction method:
Determine the target site of the gene to be knocked out;
Design to identify the target sites from a pair of sgRNA;
Construct Cas9 plasmid that expresses sgRNA;
In vitro transcription of sgRNA and Cas9 RNA;
The sgRNA and Cas9 RNA are injected directly into the fertilized eggs to detect sgRNA activity;
The active sgRNA and Cas9 RNA are injected directly into the fertilized eggs to establish Founder;
Founder will be F1 generation after self-fertilization.
Application:
Preparation of knockout mice: knockout mice can be used for gene function research; for gene knockout in cells, you can choose lentiviral vector to construct lenticrispr-v2 plasmid, through packaging virus for cell knockout.
2. Gene knockdown
RNA level refers to the ability to prevent gene expression by degrading mRNA having a homologous sequence target gene. It is generally used for cell level knockdown genes.
A.RNA interference:
SiRNA double-strand binds to a ribozyme complex to form a so-called RNA-induced silencing complex (RISC). The activated RISC is mapped to the homologous mRNA transcript by base pairing and cleaves mRNA at a position of 12 bases from the siRNA 3’ end, which is degraded to cause gene expression silencea—a specific post-transcriptional gene silence.
The synthesized siRNA for the target gene is generally transfected into the cell by transfection to silence the target gene. This method is greatly affected by transfection efficiency. Nowadays, there are many commercial genetic siRNA products, so it is very convenient.
Application:
After synthesis, it usually performs cell level knockdown by transfected cells.
B. shRNA
The shRNA consists of two short reverse repeats. The central section separated by a loop sequence, consisting of a hairpin structure, is controlled by the pol III promoter. Then 5-6 T is followed by as a transcription terminator for RNA polymerase III.
Construction of shRNA viral expression vector commonly uses adenovirus vector, adeno-associated virus vector and lentiviral vector. And the mechanism is similar to that of plasmid vector, by using characteristics that virus infection cell efficiency is relatively high to solve the defects that plasmid vector transfection is low in efficiency.
Construction method:
ShRNA design: designing RNA interference sequences is the key to RNAi experiments;
Vector construction: choosing lentivirus or adenovirus vector according to the experimental needs;
Transfected cells: commonly used methods include calcium phosphate transfection, liposome transfection, electroporation, etc.;
Observation of GFP Fluorescence and Stable Expression Cell Lines Screening: it can be used for plasmid vectors with GFP tags;
Detection of RNA interference efficiency: RNA efficiency can be detected at protein level or mRNA level.
Application:
Generally, it performs cell-level knockdown by building lentivirus, adenovirus and others.
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