Innovative CRISPR Enzymes Outsmart Immune Defenses: A Breakthrough in Gene Editing

The fundamental elements of CRISPR-based genomic editing therapies are bacterial proteins referred to as nucleases, which can incite unintended immune responses in individuals, raising the likelihood of adverse effects and potentially decreasing the efficacy of these treatments.

Scientists at the Broad Institute of MIT and Harvard, along with Cyrus Biotechnology, have recently modified two CRISPR nucleases, Cas9 and Cas12, to conceal them from the immune system. The research team pinpointed protein sequences on each nuclease that activate the immune system and applied computational modeling to craft new variants that avoid immune detection. The altered enzymes demonstrated comparable gene-editing effectiveness and diminished immune responses compared to conventional nucleases in mice.

Published today in Nature Communications, the outcomes may facilitate the development of safer, more effective gene therapies. The investigation was spearheaded by Feng Zhang, a core institute member at the Broad and an Investigator at the McGovern Institute for Brain Research at MIT.

“As CRISPR therapies progress into clinical settings, there is an escalating necessity to verify that these tools are as secure as possible, and this effort addresses one aspect of that challenge,” remarked Zhang, who also serves as a co-director of the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics, the James and Patricia Poitras Professor of Neuroscience, and is a faculty member at MIT. He is an Investigator at the Howard Hughes Medical Institute.

Rumya Raghavan, a graduate student in Zhang’s laboratory at the study’s onset, along with Mirco Julian Friedrich, a postdoctoral researcher in Zhang’s lab, were co-first authors of this research.

“It has been known for some time that Cas9 elicits an immune response, yet we aimed to identify the specific components of the protein recognized by the immune system and subsequently manipulate the proteins to eliminate those parts while preserving their function,” explained Raghavan.

“Our objective was to utilize this knowledge to devise not only a safer therapy but one that could potentially be even more effective by not being deactivated by the immune system before fulfilling its role,” Friedrich added.

In pursuit of immune triggers

A multitude of CRISPR-based therapies utilize nucleases derived from bacteria. Approximately 80% of individuals possess pre-existing immunity to these proteins due to regular exposure to these bacteria, although scientists were unaware of which segments of the nucleases the immune system acknowledged.

To investigate, Zhang’s team employed a specialized variety of mass spectrometry to detect and scrutinize the Cas9 and Cas12 protein fragments recognized by immune cells. For both nucleases—Cas9 from Streptococcus pyogenes and Cas12 from Staphylococcus aureus—they identified three brief sequences, roughly eight amino acids in length, that triggered an immune response.

They subsequently collaborated with Cyrus Biotechnology, a firm co-established by University of Washington biochemist David Baker that develops structure-based computational tools to create proteins that avoid immune reactions. Following the identification of immunogenic sequences in Cas9 and Cas12 by Zhang’s team, Cyrus employed these computational strategies to devise variations of the nucleases that excluded the immune-triggering sequences.

Zhang’s laboratory utilized predictive software to confirm that the novel nucleases were less probable to elicit immune responses. Subsequently, the team engineered a suite of new nucleases based on these predictions and evaluated the most promising candidates in human cells as well as in mice genetically modified to incorporate crucial components of the human immune system.

In both tests, they discovered that the engineered enzymes yielded significantly lower immune responses compared to the original nucleases, while still excising DNA with equivalent effectiveness.

Minimally immunogenic nucleases form just one aspect of safer gene therapies, according to Zhang’s team. Looking ahead, they aspire that their approaches might also aid scientists in devising delivery mechanisms that can evade the immune system.