A brief introduction of gene editing techniques CRISPR/Cas9 and Argonaute
The development of science and technology makes people to know the disease deeply. It was found that the etiology of many human diseases has an inseparable relationship with gene mutation. So, how to treat these diseases? People naturally think of re-editing mutant gene by using some techniques, this technology is gene editing technique. Based on protein/DNA identification and cut, gene editing techniques have been through the first generation of gene editing techniques, such as homing endonuclease, ZFN and TALEN. With the constantly understanding of the gene editing techniques, people realized the first generation of gene editing techniques have many defects, such as fussy operation, low efficiency, serious phenomenon of off-target, so it is hard to be applied in production practice. In 2003, MIT team first used prokaryotic CRISPR/Cas9 system to successfully edit eukaryotic genes, and this technique had simple operation, high efficiency and less-severe off-target. The second generation of gene editing techniques, represented by CRISPR/Cas9 system, is based on RNA & DNA recognition and protein mediated cut. In 2016, Han chunyu team used prokaryotic Argonaute system to successfully edit eukaryotic genes. Compared with CRISPR/Cas9, this technique has simpler operation, higher efficiency and lesser-severe off-target, in addition, this technique is not harsh to the genes location in chromosome. This technique is likely to be the third generation of gene editing techniques, which is based on DNA & DNA recognition and protein mediated cut. In the following sections, we will provide more details on gene editing techniques of CRISPR/Cas9 and Argonaute system.
1.CRISPR/Cas9 genome editing technique.
CRISPR/Cas9 exists widely in prokaryote and consists of coding region of Cas9, a guide RNA and CRISPR cluster. CRISPR cluster contains discrete repetitive sequence R and intergenic spacer sequence S, R and S are intervally arranged. In prokaryotes, exogenous DNA is inserted into the genome, once transcription start, organisms will cut the sequence into pieces which is near to PAM (NGG sequence), this short sequence is added to CRISPR cluster. When the same exogenous DNA is inserted again, Cas9 will combine with short RNA sequence (guide RNA) which is transcribed by CRISPR cluster, the complex will be transported to nucleus and recognize PAM to scan the genome-wide. When the complementary DNA against RNA is appeared, it will be spliced by Cas9, the invasion of exogenous DNA is blocked. Based on this, we can wrap up CRISPR/Cas9 system in adenovirus or lentivirus, then the virus invade mammalian cells, the splicing site is recognized according to the sequences inserted into CRISPR cluster(the sequences is entirely 19 bases matching genome at least ), then the fixed shear of eukaryotic cell genome is finished. After cutting, double-strand breaks (DBS) is showed up in target site, the repair of organism is triggered under a certain condition (HDR (Homology directed repair) and NHEJ (Non-homologous end joining)).
The experimental process of gene knock-out based on CRISPR/Cas9 system: 1. Construct plasmid of Cas9 gene with nuclear localization sequence (NLS) and another constructed plasmids which can produce guide RNA (design in advance); 2. The virus packing, the titer test; 3. Infection of mammalian cells; 4. The effect of gene knocking was detested by ELISA, WB and sQRT-PCR.
2.Genome editing techniques using the Argonaute system.
Argonaute system consists of coding region of Argonaute and short sequences of double-stranded DNA with 5' phosphorylation, found in only a few prokaryotes. ssDNA with 5' phosphorylation does not exist in eukaryotes cytoplasmic matrix, Argonaute is invaded into mammalian cells by adenovirus or lentivirus packaging, lipofection transfection and electric shocking. While ssDNA with 5 'phosphorylation is transferred to cell by artificial synthesis. Argonaute expressed in cells will specifically combine ssDNA with 5' phosphorylation, the complex will be transported to nucleus, and the splicing site is recognized by the ssDNA (the optimum sequences is entirely 24 bases matching genome), then the fixed shear of Eukaryotic cell genome is finished. After cutting, double-strand breaks (DBS) is showed up in target site, the repair of organism is triggered under a certain condition.
The experimental process of gene knock-out based on Argonaute system: 1.Construct plasmid of Argonaute gene with nuclear localization sequence (NLS) and artificially synthetic ssDNA (design in advanced); 2. Plamid and ssDNA is transfected with lipofectine to mammalian cells (also works with electric shock); 3. The effect of gene knocking was detected by ELISA, WB and sQRT-PCR.
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