IBS scientists have discovered the smallest CRISPR-Cas9 family members so far, studies have shown that it can edit mutations by gland-associated viruses to blinding genes.
Researchers at the Institute for Basic Science (IBS), Seoul, Korea, etc. designed the smallest CRISPR-Cas9 and delivered it to myocytes and mice via adeno-associated virus (AAV) Eyes, used to edit the genes that lead to blindness.
This study was published on Nature Communications, a CRISPR-Cas9 system derived from Campylobacter jejuni (CjCas9), which may become a new tool for effective treatment of general disease and "disease-free" diseases.
CRISPR-Cas9 is red, and as a "gene scissors" protein, Cas9 needs to be cleaved at the exact target gene position under guided RNA guidance. The CRISPR-Cas9 complex needs to be delivered to the target DNA by plasmid or viral delivery. "AAV is an effective and safe carrier for expressing the target gene in vivo, and has been widely used in gene therapy," explains KIM Jin Soo, director of the IBS Genome Engineering Center.
In natural conditions, Cas9 is a bacterial immune weapon that cleaves DNA that can damage bacteria. The most common CRISPR-Cas9 technology uses Cas9 from Streptococcus procephalus Strain. However, this protein consists of 1,368 amino acids, too much volume can not be packaged and delivered by AAV. Even if the scientists will be divided into two parts, were packaged in different viruses, there will be other problems, such as the need for twice the amount of virus, the activity is smaller than the complete Cas9. Staphylococcus aureus Cas9 can also be used for gene editing and is slightly smaller (1,053 amino acids), so it can be transported by AAV, but this Cas9 does not have enough space to load other proteins.
In this latest study, the researchers found that CjCas9 was sized and efficient, and that the protein had 984 amino acids that could be packaged into AAVs with multiple directed RNAs and fluorescent reporter genes.
In order to use bacterial proteins for gene editing, the researchers optimized the technique by designing a short DNA sequence that follows the DNA sequence immediately adjacent to the Cas9 target, the Protospacer Adjacent Motif (PAM). Each of the different Cas9 requires a specific PAM sequence, otherwise it can not be combined and cleaves the target DNA sequence. In addition, the researchers also modified the length of the directed RNA.
Following this optimization, the researchers packaged the new CRISPR-Cas9 complex, along with two directed RNAs and fluorescent reporter proteins, into AAV, toward the mutant gene in mouse muscle and eyes. They focus primarily on two genes involved in age-related macular degeneration (AMD), which is one of the main causes of adult blindness.
A gene is a common therapeutic target for ADM, known as vascular endothelial growth factor A (VEGF A), and the other gene is a transcription factor that activates transcription of VEGF A: HIF-1a. The latter had not previously been used as a drug target. In this study, the team demonstrated that CjCas9 was delivered to the retina through AAV and was effective in inactivating Hif-1a and VEGF A in mice and reducing the area of choroidal neovascularization (CNV).
"CjCas9 is highly specific and does not exhibit off-target mutations in the genome," explains KIM Jin-Soo.
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