This is an adeno-associated virus containing the DNA coding for the RNA guide, the Cas9 protein derived from Campylobacter jejuni, and the green fluorescent reporter protein (GFP). This is possible because of the small size of Cas9. [IBS]

Being able to package the genome-editing tool CRISPR/Cas9 into a delivery system that can readily target a vast number of tissues in the body has been the goal of molecular biologists since the editing technique as discovered a few years ago. Now, scientists at the Center for Genome Engineering, within the Institute for Basic Science (IBS) in Korea, in collaboration with ToolGen, and Seoul National University have engineered the smallest CRISPR/Cas9 to date, delivered it to the muscle cells and in the eyes of mice via adeno-associated viruses (AAV), and used it to modify a gene causing blindness. The new, small Cas9 enzyme, expected to become a useful therapeutic tool against common and undruggable disease targets, originated from the bacteria Campylobacter jejuni—a common cause of food poisoning.

The findings from this study were published recently in Nature Communications in an article entitled “In Vivo Genome Editing with a Small Cas9 Orthologue Derived from Campylobacter jejuni.”

The CRISPR/Cas9 system has ignited imaginations within the molecular biology field as a real source of molecular therapy for a host of diseases. The technique is an innovative, cheap, and precise technique to edit genes—working as a pair of molecular scissors to create cuts on the target gene in precise locations indicated by guide RNA. Yet, for the CRISPR/Cas9 complex to reach its target DNA, it has to be delivered via plasmids or viruses.

“AAV is an efficient and safe vector to express a gene of interest in vivo and has been used widely in gene therapy,” explained co-senior study investigator Jin-Soo Kim, Ph.D., director of the IBS Center for Genome Engineering.

The Cas9 enzyme is used by several bacteria as an immunity weapon—it is needed to cut viral DNA that could damage the bacteria. The most common version of the CRISPR/Cas9 technique uses Cas9 derived from the bacterium Streptococcus pyogenes. However, this protein is made of 1368 amino acids, and it is too large to be delivered and packaged in AAV. Even if scientists split it up into two parts, each packaged in a different virus, other issues arise. A double amount of viruses need to be delivered, and the split Cas9 is less active than the intact SpCas9. Over the years researchers have discovered smaller versions of the Cas9 enzyme, such as the one derived from Staphylococcus aureus (1053 amino acids). However, while this version can just fit inside the AAV, it does not leave enough space for other essential proteins.

In the current study, the research team found that C. jejuni Cas9 is both efficient and small. At 984 amino acids, it can be packed into AAV together with multiple guide RNAs, as well as a fluorescent reporter protein.

Streptococcus pyogenes, Staphylococcus aureus, and Campylobacter jejuni. The Cas9 protein derived from C. jejuni has only 984 amino acids (aa), and it is the smallest one developed for gene editing so far. [IBS]” width=”60%” height=”60%” />



“We present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo,” the authors wrote. “After determining protospacer-adjacent motif (PAM) sequences and optimizing single guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome.”

To use a bacterial protein for gene editing, the investigators had to optimize some aspects of the technique. They designed a short DNA sequence immediately following the sequence targeted by the Cas9, called the protospacer adjacent motif (PAM). Each different Cas9 needs a specific PAM sequence, otherwise, it will not be able to bind to and cleave the target DNA sequence. Additionally, they had to modify the length of the guide RNA.

Once their modifications were complete, the researchers packaged the new CRISPR/Cas9 complex into AAV, together with two guide RNAs and a fluorescent reporter protein, to mutate genes in mouse muscles and eyes. The scientists focused on two genes involved in the age-related macular degeneration (AMD), one of the leading causes of blindness in adults. One gene is a common therapeutic target for ADM, called vascular endothelial growth factor A (Vegfa), the other one is a transcription factor that activates the transcription of Vegfa and it is known as Hif1a.

“CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells,” the authors penned. “Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.”

Intraocular injections of AAV-packaged CRISPR/CjCas9 could be beneficial to treat various retinal diseases and systemic diseases “CjCas9 is highly specific and does not cause off-target mutations in the genome,” noted Dr. Kim.

Hif1a gene target sequences are the same in both mice and humans; thereby the method presented in this study could be used in the future for the treatment of ADM in human patients. By paving the way to the application of CjCas9 against undruggable genes or noncoding sequences, this technology can broaden the range of therapeutic targets, making the entire human genome potentially druggable.























