Nat Commun: the smallest gene scissors

The Institute of Basic Sciences (IBS) in collaboration with KIM Eunji (ToolGen Inc.) and KIM Jeong Hun (Seoul National University) designed the smallest CRISPR-Cas9 to date and delivered it to myocytes and mouse eyes through adeno-associated virus (AAV) and used it to modify the blinding genes. This CRISPR-Cas9 system is derived from Campylobacter jejuni (CjCas9) and is expected to be an effective "non-exclusion" disease treatment tool. The study has been published in Nature Communications, which also publishes other studies on recombinant mouse proteins.

In this study, the team found that CjCas9 was both efficient and small enough. It has 984 amino acids, which can be packaged in AAV with multiple directed RNAs and fluorescent reporter proteins. In order to use bacterial proteins for gene editing, scientists had to optimize some aspects of the technology. They designed a short DNA sequence followed by targeting the DNA sequence by Cas9, known as the Protospacer Adjacent Motif (PAM). Each of the different Cas9 requires a specific PAM sequence; otherwise the target DNA sequence will not be bound and cleaved. Secondly, they had to modify the length of the wizard RNA.

Subsequently, IBS scientists packaged the new CRISPR-Cas9 complex with two guide RNAs and fluorescent reporter proteins into AAV to modify the genes in mouse muscle and eyes. They focused on two genes involved in age-related macular degeneration (AMD), because these two genes are 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 VEGF A transcription, known as HIF-1a. Unlike VEGF A, HIF-1a is not considered a drug target. The so-called "miscarriage" genes, such as the usual transcription factors, can't be targeted directly by antibodies and other biological or chemical agents. In this study, the team demonstrated that CjCas9 was efficiently delivered to the retina through AAV to inactivate Hif1a and VEGF A in mice and reduce the area of choroidal neovascularization (CNV).

Intraocular injection of AAV-packaged CRISPR-CjCas9 can be beneficial in the treatment of various retinal diseases and systemic diseases. KIM Jin-Soo explains that CjCas9 is highly specific and does not cause off-target mutations in the genome.

The mouse and human Hif1a gene target sequences are identical, so the method proposed in this study will be useful in the treatment of ADM in human patients. By paving the way in which CjCas9 is applied to "unavoidable" genes or non-coding sequences, this technology broadens the scope of therapeutic targets so that the entire human genome is potentially drugizable. By the way, Flarebio offers recombinant proteins of good quality like recombinant COLEC12.