The CRISPR-Cas9 protein from wild-type Staphylococcus aureus (SaCas9) is a diamond in the rough. The protein has potential because it is small—small enough to fit inside the adeno-associated virus (AAV), a vector commonly used to deliver genome editing components to living cells. Wild-type SaCas9, however, typically lacks high-genome-wide specificity. Consequently, a little cutting and polishing is needed if SaCas9 is to shine in genome editing applications.

SaCas9 underwent cutting and polishing—that is, rational engineering—at the Ming Wai Lau Center for Reparative Medicine, a branch of the Karolinska Institutet in Hong Kong. This work, which was carried out by a scientific team led by Zongli Zheng, PhD, an assistant professor at the Karolinska Institutet, resulted in an SaCas9 variant called SaCas9-HF that is significantly more accurate than wild-type SaCas9.

“[Our variant] provides an alternative to the wild-type Cas9 toolbox—a new SaCas9 where highly precise genome editing is needed,” said Zheng. “The new enzyme will be particularly useful for future gene therapy using AAV to deliver genome editing components in vivo.”

Detailed findings appeared September 30 in the Proceedings of the National Academy of Sciences, in an article titled, “Rationally engineered Staphylococcus aureus Cas9 nucleases with high genome-wide specificity.” This article not only describes how the SaCas9-HF variant was engineered, it also describes the variant’s performance, as determined by the GUIDE-seq (genome-wide unbiased identification of double-stranded breaks enabled by sequencing) method and targeted deep sequencing analyses.

“Among 15 tested human endogenous sites with the canonical NNGRRT protospacer adjacent motif (PAM), SaCas9-HF rendered no detectable off-target activities at nine sites, minimal off-target activities at six sites, and comparable on-target efficiencies to those of wild-type SaCas9,” the article’s authors wrote. “Furthermore, among four known promiscuous targeting sites, SaCas9-HF profoundly reduced off-target activities compared with wild type.”

According to the article, SaCas9 variants with highly specific genome-wide activity in human cells could be rationally engineered without compromising on-target efficiency.

Zheng and colleagues also considered how well SaCas9-HF performed after it was delivered by an AAV. SaCas9-HF, the scientists reported, showed reduced off-target effects when targeting VEGFA in a human retinal pigmented epithelium cell line compared with wild type.

SaCas9 variants are of interest because some versions of Cas9, such as the Cas9 nuclease from Streptococcus pyogenes (SpCas9), has a high target precision but can be too large for the viral particle needed to deliver the gene editing kit to the cells. Although SaCas9 is much smaller than SpCas9 and can easily fit into the AAV vector, SaCas9’s wild-type variant lacks SpCas9’s high precision. Imprecise gene-editing can end up modifying DNA at unintended places with potentially serious consequences.

More research on additional target sites and cell types will be needed to determine if the SaCas9-HF variant is equally precise across different cell types, although the researchers say they are optimistic about its application across many other cell types.

In terms of how good the enzyme was in modifying the intended genomic locations, the SaCas9-HF had an average on-target efficiency of 80% compared to the wild type, which is generally considered comparable in the current field, according to the researchers. In some CRISPR-Cas9 cases, a 10% editing efficiency is enough to repair the damaged gene sequence and restore function.

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