NEW YORK (GenomeWeb) – A team of Korean researchers has engineered what they say is the smallest Cas9 ortholog to date.
Cas9 is a key component of the CRISPR gene-editing tool — the first and most commonly used Cas9, derived from Streptococcus pyrogenes (SpCas9),is some 1,370 amino acids large. According to researchers from the Institute for Basic Science in South Korea, that is too bulky to fit into certain viral vectors for delivery, and breaking it up into two vectors makes it less active. While the Staphylococcus aureus Cas9 (SaCas9) is smaller and fits into vectors, it doesn't leave much room for reporter and other genes.
To solve the problem, the IBS researchers and their partners at Seoul National University Hospital and biotechnology company ToolGen derived a Cas9 ortholog from Campylobacter jejuni (CjCas9) that was even smaller than SaCas9, coming in at 984 amino acids. As they reported in Nature Communications this week, they also packaged it into adeno-associated virus vectors and delivered it to mouse muscle and retinal cells, suggesting it could be used to target genes previously thought to be undruggable.
"CjCas9 is highly specific and does not cause off-target mutations in the genome," senior author Jin-Soo Kim from IBS said in a statement.
In their paper, the researchers noted that CjCas9 designed to target six different DNA regions cleaved between one and 27 sites in the human and mouse genomes, suggesting that it is specific. By comparison, SpCas9 cleaved some 15 to 147 sites.
Kim and his colleagues further noted that this higher CjCas9 specificity did not appear to come at the expense of a lower editing efficiency. They suggested that its increased specificity could be in part due to its optimal single-guide RNA (sgRNA) length of 22 nucleotides and four nucleotide protospacer-adjacent motif sequence being slightly longer than those for SpCas9.
Likewise, in a head-to-head comparison, they found CjCas9 to be as efficient as SaCas9, though more specific.
Kim and his colleagues packaged CjCas9 into AAV vectors. For one, they directed it at the Rosa26 locus in C2C12 mouse myotubes to find that indels accumulated in a time- and dose-dependent manner. They also bundled it with a muscle-specific promoter and Rosa26-specific guide RNA and injected it into mouse muscles to find indels at the sites with a frequency of about 17 percent after eight weeks and 13 percent after 32 weeks. This suggested to the researchers that long-term expression of CjCas9 might not lead to increased off-target effects.
They also tested whether CjCas9 could be used in the eye. They reported that CjCas9 could induce indels at Rosa26, Vegfa, and Hif1 target sites in mouse retinas. In addition, in a model of age-related macular degeneration, they noted that CjCas9 targeted to the Vegfa and Hif1 sites reduced the size of laser-induced choroidal neovascularization.
VEGF A, a secretory protein, is a target of AMD drugs, while HIF-1alpha activates VEGF A, but has been thought to undruggable as it cannot be targeted by antibodies or small molecules, the researchers noted.
"Because the CjCas9 target site in the mouse Hif1a gene is perfectly conserved in the human HIF1A gene, the AAV presented in this study or its variants could be used for the treatment of human patients in the future," Kim and his colleagues wrote in their paper. "We expect that CjCas9 can be directed to other traditionally 'undruggable' genes or non-coding sequences to broaden the range of therapeutic targets, making the entire human genome potentially druggable. "