The researchers profiled six small Cas9 orthologues, which can edit genomes the same way, but are more than one kilobase shorter.
Gene editing in animals using CRISPR-Cas9 has been limited by the size of the Cas9 protein.
Cas9, derived from Streptococcus pyogenes (SpCas9) is too big for in vivo research and therapeutic applications, even those using an adeno-associated virus (AAV) delivery vehicle.
“The restrictive cargo size (4.5 kilobases, excluding the inverted terminal repeats) of AAV presents an obstacle for packaging the commonly used Streptococcus pyogenes Cas9 (SpCas9, 4.2 kb) and its single guide RNA (sgRNA) in a single vector,” said authors of a paper from the Broad Institute, including Feng Zhang, the discoverer of CRISPR-Cas9 editing.
Although technically editing is possible using SpCas9 and an AAV vector, the approach makes customised expression very difficult.
The scientists characterised six Cas9 enzymes with particularly short receiver domains – Cas9 from Staphylococcus aureus (SaCas9), belonging to Type IIA and IIC subtypes.
They then performed in vivo tests of SaCas9 and its sgRNA with an AAV vector in mice.
“We packaged SaCas9 and its single guide RNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver,” said the scientists.
The dose has the intended therapeutic effect: “Within one week of injection, we observed .40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels.”
The findings could have clinical potential: PCSK9 inhibitors are a class of cardioprotective drugs, with studies in human associating loss of PCSK9 with a reduced risk of cardio disease and lower cholesterol. They also open up potential for other gene therapies using a combination of SaCas9 and AAV delivery.
The researchers also assessed the targeting specificity of SaCas9 and SpCas9 using a BLESS assay (direct in situ breaks labelling, enrichment on streptavidin and next-generation sequencing) which identifies off-target cleavage sites in a mouse cell line. They concluded in vivo genome editing using SaCas9 “has the potential to be efficient and specific” with “tremendous promise for biomedical research.”
“Identification of new Cas9 orthologues, in addition to structure-guided engineering, could yield a repertoire of Cas9 variants with expanded capabilities and minimized molecular weight, for nucleic acid manipulation to further advance genome and epigenome engineering.”