Exciting Cas9 new usage: Synergistic Activation Mediator
The CRISPR/CAS9 technology is very familiar to everyone and has become the mainstream gene editing technology. If you want to send high scores, SCI articles don't use CAS9 to knock out the genes and are embarrassed to vote for the magazine. As a sensational technology in the biosphere, this technology invented by Dr. Zhang Feng of the Chinese (please forgive me for automatically ignoring Dr. Doudna) has made progress recently.
To know the function of a gene, its research methods are nothing more than Loss-of-function (LOF) and gain-of-function (GOF). Gene function can be studied by LOF using both RNAI and CAS9 techniques, and both methods can construct a complete target library for high-throughput gene function screening. However, life without LOF without GOF is incomplete. GOF requires us to construct a gene CDS region into a vector and overexpress the gene in the cell. However, most genes have many transcripts, and they cannot be designed for public areas like RNAI or CAS9. They can only be constructed one by one, and many huge genes are difficult to construct into vectors. These factors limit the research of many researchers on the function of low-expression genes. These students don't have to worry anymore, and a new technological innovation has brought new hope to the topic that you may be heading to a dead end.
Synergistic Activation Mediator is referred to as SAM. He is a new technology that uses the CAS9 method to overexpress endogenous genes. You are not mistaken, it is expressed in CAS9! Let's take a look at his technical principles.
In fact, Dr. Zhang Feng has developed a mutant CAS9 protein dCAS9 that binds only to the DNA sequence without cleavage. After fusion with dCAS9 and the transcriptional activator VP64, it can specifically localize to the promoter region of the gene and activate its transcription.
However, the excellent scientists are all "pretty". In order to more effectively express the endogenous genes, a series of transformations have been started, and finally we have obtained the SAM system we have seen.
Let's take a closer look at the components of the system:
VP64
Is a transcriptional activator that recruits transcription factors like PC4, CBP/p300 and SWI/SNF complexes.
sgRNA2.0
sgRNA2.0 is different from traditional sgRNA in that it has two MS2 protein binding positions (red neck ring)
MS2-p65-HSF1
It is a protein produced by the fusion of three genes, in which MS2 is used to bind p65, HSF1 and sgRNA, and p65 and HSF1 are also VP64-like transcriptional activators, which can recruit AP-1, ATF/CREB, SP1, etc. Transcription factor
When these components are combined, they can exert the over-expression effect of the blasting day.
The endogenous overexpression of the two genes, ASCL1 and MYOD1, has reached hundreds of times, and has been similar to the method of overexpression of cDNA.
Let's open up the SAM to do what it can do. First of all, because the gene is too big to be over-expressed, the students crying in the corner finally got the weapon. In addition, do you often cry to the NCBI page, why do I have to express so many transcripts! Don't be afraid now, use SAM to control how many transcripts you have, as long as it is a promoter to start all. Students who have expressed high-throughput screening can also use the uniform format of the SAM target library for large-scale overexpression studies, and moms no longer have to worry about what I have said.
And on the basis of SAM technology, other "pretty" scientists have developed a light-controlled over-expression system. Fusion of blue-activated CIB1 and CRY2 genes on dCAS9, linked to a transcriptional activator P65, when illuminated with blue light (for GFP), the gene can be overexpressed to overexpress as a controlled process in vivo Level is a very meaningful technical improvement. 
The Jikai gene has been able to provide SAM and light-activated SAM viruses! Comrades who have expressed their opinions, what are you waiting for, COME ON! The era that belongs to you is here!
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