CRISPR is a kind of immune mechanism from invading viruses DNA or other exogenous DNA. In bacteria and archaebacteria fields, CRISPR system can be divided into 3 classes, class I and class III needs a variety of CRISPR related protein (Cas protein) to play a role, and class only needs Cas protein, which is widely used to provide convenient conditions.
At present, the application of CRISPR-Cas9 system from Streptococcuspyogenes is the most wide one.Cas9 protein contains two nuclease enzyme domains which can cut two DNA single strands.Cas9 first combines with crRNA and tracrRNA, and then goes through PAM sequence to get into DNA, forming RNA-DNA complex structure, so that cut the DNA double stands.
Because the PAM sequence has the advantages of simple structure. A large number of targets can be found almost in all genes. So it has been widely used.CRISPR-Cas9 system has been successfully applied to the plant, bacteria, yeast, fish and mammalian cells. It is currently the most efficient genome editing system.
By means of genetic engineering, sgRNA gained by the improvement of crRNA and tracrRNA which are connected together.RNA fused has the same activity with the wild type RNA, but because its structure is so simple so it is more convenient for users. By linking the originals of expressed sgRNA and Cas9,there can obtained plasmids expressing them two at the same time and the plasmids will be able to operate on the target gene.
Now the most common method to study the Cas9 is through plasmids. Although common plasmids also can achieve the effect mostly, but plasmid transfection has the disadvantages of low efficiency and short action duration. Virus appearance solves the problems of common plasmids. Common virus are lentivirus and adenovirus. Lentivirus, commonly used addgene (lentiCRISPRv2, lentiGuide-Puro, lentiCas9-Blast).It can be integrated into the host genome, expressing in a long-term stable way. But because of the slow virus clone ability and the large CAS9 molecular weight (more than 4KB), lower titer and the disadvantages that long insertion may lead to chaos, miss, adenovirus has more advantages. Adenovirus has a strong cloning ability. And when taking into use, it effects for a long time compared with normal plasmids, and can achieve more ideal knockout effect in the end.
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