Before CRISPR-Cas9 can be used with confidence in the human body, it must demonstrate that it is safe—and not just with respect to off-target effects, which preoccupy many scientists adapting CRISPR-Cas9 to edit genes in cells in human tissues. Another dimension of safety is immunogenicity toward the Cas9 protein, which is, after all, a foreign body.

To confront the immunogenicity problem head on, scientists based at Arizona State University (ASU) have identified the binding sites or epitopes on Cas9 that attract the attention of the immune system’s T cells. The scientists, led by Karen S. Anderson, PhD, a professor at the Biodesign Institute at ASU’s Virginia G. Piper Center for Personalized Diagnostics and ASU’s School of Life Sciences, also engineered a modified version of Cas9 that lacks these epitopes. The modified Cas9, then, is an incognito version of the usual form of Cas9, which comes from Streptococcus pyogenes, a bacterium to which many people have already developed immunity.

So, is the incognito version of Cas9 effective? According to the ASU team, it retained its DNA-cutting ability while reducing T-cell reactivity. In fact, T-cell reactivity showed a 25–30-fold reduction.

Detailed findings appeared April 23 in Nature Communications, in an article titled, “Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes.” The article presents evidence that the Cas9 protein can be modified to “eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity.”

The article affirms that Cas9 is indeed immunogenic in humans and that preexisting exposure to S. pyogenes can drive the body’s T cells to launch an immune attack against the bacterial protein. When 143 samples of blood were screened, 82 of them (or 57.3%) showed detectable levels of antibody to S. pyogenes.

The study also describes a fully functional version of Cas9, suitable for gene editing, that is not recognized and targeted by the immune system. To do generate this undercover Cas9, the researchers identified the regions of antibody binding on the Cas9 molecule (known as epitopes) that were directly implicated in triggering T cell recognition and attack.

Two mutations in so-called anchor residues of the Cas9 epitope were explored individually and in combination to assess their effect on immunogenicity. Modifying these regions by just a single amino acid produced a version of Cas9 that could operate undercover.

“That’s the unique part of what we’ve done,” Anderson said. “We took those dominant epitopes and tried to silence them—just by doing one or two mutations in the Cas9 gene. But we rebuilt it, so the gene was still functional. It’s not immunologically silent, but it’s more quiet.”

Indeed, the study results confirmed that in cultured cells, the reengineered Cas9 was less immunologically active, while retaining its functional properties. The authors stress that the technique could be combined with other strategies to further improve CRISPR safety and reduce the need for immunosuppressant drugs.

In previous gene editing efforts, cells were removed from human tissue, reengineered, and replaced in the body. The power of CRISPR allows researchers to modify DNA within a living person’s tissue and even to target multiple gene modifications with a single CRISPR intervention.

“If you want to think about repairing cells that are in an organ, like a liver cell or kidney or brain,” Anderson pointed out, “then you have to express the bacterial protein there.” This is where the threat of triggering an immune response to Cas9 becomes a formidable obstacle.

Exciting new avenues of research are being explored that would enable CRISPR to be used to induce epigenetic changes, turning on silent genes, altering the activity of disrupted genes or otherwise modifying gene expression without permanent changes to the DNA. Such interventions will require the CRISPR system to remain much longer in the body to be effective, perhaps weeks or months. Here, potential immunity to Cas9 will be even more of a critical consideration. Custom tailoring of epitopes to silence the immune response to Cas9 offers an attractive approach.

“Nonspecific localized immune suppressive approaches, such as those used by tumor cells and some viruses may complement these strategies for complete deimmunization,” the authors of the current article noted. “Deimmunized Cas9 may be useful in reduction of the dosage of other immunomodulatory measures needed to be co-administered in patients, thus facilitating therapeutic CRISPR applications as we develop a better understanding of the immunological consequences of this system.”

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