CRISPR/Cas technology is the latest progress:keep the DNA intact and also activation of target genes

Source: CRISPR/Cas technology is the latest progress:keep the DNA intact and also activation of target genes

gene-editing technology CRISPR/Cas system because of its easy operation, energy efficient and precise at specific sites on the DNA to be cut and edited, in recent years has been in the field of biotechnology research hotspot. CRISPR/Cas is the bacteria and archaea in the long-term evolution process of the formation of an adaptive immune defense mechanisms to fight viruses and foreign DNA invasion. Wherein the Cas (CRISPR associated protein)is a kind of endonuclease, and containing the target DNA sequence complementary to the guide RNA under the guidance of identify DNA specific sites and cut, forming a double-stranded DNA gap, and then the cells by means of homologous recombination or non-homologous end joining mechanism to break DNA repair, thereby forming a DNA insert or remove mutations, to achieve gene editing.

RNA-mediated CRISPR/Cas9 system for targeted genome modification mechanism of action diagram

although based on the CRISPR gene editing system, in many human disease in a mouse model shows great therapeutic potential, but Cas9-induced DNA Double-Strand breaks(DNA double-strand breaks, and DSBs)of the process may be off-target, the introduction of unknown mutations, the patient caused by injury, limiting its clinical application.

improvements to CRISPR/Cas9 technology

The Nature Reviews Genetics senior editor Darren J. Burgess the recent author of“Translational genetics: the CRISPR therapies – making the grade not the cut”, the reviews of the U.S. Salk Institute for biological Juan Carlos IzpisuaBelmonte Research Group researchers use to improve the CRISPR/Cas9 technology-mediated transcription-epigenetic regulation in mice in vivo activation of multiple target genes work.

Juan Carlos Izpisua Belmonte, the research group improved the CRISPR/Cas9 system shown

use the CRISPR system for transcription regulation, usually does not have nuclease cutting function Cas9 (dCas9)and can regulate the transcription of a region of the protein fusion, this region of the protein may be a transcription activation factor, transcription inhibition factor or histone modification enzyme. Then the fusion protein is designed a single-guide RNA (single-guide RNA, sgRNA)directed to the target gene location. Since the most suitable gene therapy vector is an adeno-associated virus (AAV), but the adeno-associated virus load capacity is limited, can not carry the typical dCas9 fusion protein encoding gene. In order to overcome the dCas9/sgRNA and synergistic transcription activation complex of the coding sequence beyond the common AAV capacity of technical problems, Juan Carlos Izpisua Belmonte of the research group the researchers of the CRISPR/Cas9 system has been improved.

single-guide RNA(sgRNA)improvement

first, they improve the sgRNA, the introduction of the MS2 loop, so that it does not require the Cas9 fusion, in an independent way to raise a transcriptional activation protein MS2–P65–HSF1 (MPH); second, they shorten the sgRNA sequence length(14 or 15 base pairs instead of the 20 base pairs), making it even with the wild-type Cas9 combine to form a complex, the complex of the target gene to be cut(the researchers put these sgRNA called dgRNAs)。 With the above improvement, the researchers can facilitate the take based on the CRISPR transcription activation component is encoded into the 2 AAV: a coding dgRNA and the transcription activation protein MPH, another encoding Cas9, let sgRNA binding transcription-activating protein MPH auxiliary Cas9 work.

The CRISPR/Cas9 technology to improve the effect of

subsequently, the research team in multiple mouse models of disease test that whether the system is able to reduce the symptoms of the disease. In most instances, they are the first to use cell cultures to determine can effectively activate target gene dgRNAs, followed by an AAV vector will dgRNA and MPH together with the input to the expression of wild-type Cas9 transgenic mice in vivo(or by blood injection or local injection into the corresponding organs). Through experiments, they demonstrated that the upregulation of interleukin(Il10)or klotho (Kl)gene expression is able to reduce cisplatin-induced kidney injury; upregulated in the liver of the pancreatic duodenal homologous ISO-box gene 1 (pancreatic andduodenal homeobox gene 1, and Pdx1) can induce liver cells to insulin-secreting cells transdifferentiation and partial remission of streptozotocin-induced hyperglycemia (mouse Type I diabetes model); the upregulation of utrophin (Utrn)genes, whether��The newborn period or the occurrence of disease, after Duchenne muscular dystrophy(by muscle atrophy protein gene dysfunction caused by)the mdx gene models are able to alleviate the muscle weakness.

In this study, it is possible to over-expression as utrophin such large genes is striking, because the cDNA size exceeds the majority carrier of the loading capacity, by standard gene therapy method is difficult to achieve. In the expression of Cas9 in vivo in mice demonstrated upregulation of follicular inhibin(Fst) can increase muscle mass, the authors also proved that while the intramuscular injection coding dgFst–MPH and Cas9 AAV or coding dgUtrn–MPH and Cas9 AAV, without the expression of Cas9 the mdx mouse model can alleviate myasthenic symptoms.

Cas9 variants: wild-type Cas9 or dCas9

figure out the wild-type Cas9 and dCas9(i.e., does not have nuclease cutting function Cas9), which one is more suitable Cas9 variants, will be very interesting. Juan Carlos Izpisua Belmonte of the research group the research shows, in the same experiment, the wild-type Cas9 and dCas9 the Fst have the same transcriptional activation. Although the use of the Surveyor test and the depth of sequencing, they were not detected in wild-type Cas9-induced DNA Double-Strand breaks, but dCas9 does not cause DNA Double-Strand break aspect there is double insurance, if the dCas9 can be in more of the experiments proved to have a and wild-type Cas9 quite a transcriptional role, then the future dCas9 might be better variants.

after the improvements of the CRISPR/Cas9 system in mice, diabetes, muscular dystrophy and acute kidney disease in the model showed a therapeutic effect. Although we of its efficacy ability to maintain long-term there are still some questions: for example, after the first injection transcriptional regulation can last long, several times after injection, the effect whether the effect will be because the body of the AAV immune response or AAV loads of problems and gradually weakened, but this is a system for the future better for the treatment of human disease has laid a good Foundation.

summary: Juan Carlos IzpisuaBelmonte the research group the research work describes the application of the CRISPR/Cas9 activation in vivo gene therapy of the disease concept. In does not produce DNA Double-Strand breaks and maintain the DNA integrity while successfully activated target genes, regulate a physiological relevant phenotype, to avoid based on the CRISPR/Cas9 conventional gene editing technologies used in human disease treatment when there is a mutation risks. As GeorgeM. Church said, CRISPR this revolutionary technology the next stop will be the RNA-guided epigenetic regulation. (生物谷Bioon.com

Published at Mon, 07 Mar 2018 10:30:43 +0000