Harnessing the remarkable defensive mechanisms of bacteria, scientists have repurposed CRISPR, originally a bacterial immune response to viral threats, for the groundbreaking task of editing cellular genetic material. This innovative approach has recently culminated in the FDA's approval of the first CRISPR-based therapeutic for sickle cell disease in December 2023, utilizing the extensively researched CRISPR-Cas9 system.

Parallel to this, a novel CRISPR platform, dubbed Type I CRISPR or CRISPR-Cas3, is poised to revolutionize the field with its capacity for large-scale DNA removal, presenting a new frontier for therapeutic interventions.

In a pivotal study led by Yan Zhang, Ph.D., Assistant Professor in the Department of Biological Chemistry at the University of Michigan Medical School, alongside collaborators from Cornell University, significant progress has been made in improving the safety of the Type I-C/Cas3 gene editor. This research, published in the journal Molecular Cell, focuses on the development of 'off-switches' for this innovative gene editing tool. Findings from the new study were published recently in Molecular Cell.

The foundation of this research lies in the evolutionary arms race between bacteria and their viral predators, bacteriophages. The Zhang team has ingeniously crafted these CRISPR off-switches from anti-CRISPR proteins that phages have evolved to evade bacterial CRISPR immunity.

Anti-CRISPR proteins that inhibit Cas9 have previously been used in experimental settings to mitigate CRISPR's unintended effects, which occur when Cas9 interacts with non-target parts of the genome, potentially leading to undesirable outcomes, including an increased risk of cancer.

“When editing the human genome using CRISPR-Cas9, providing an inhibitor protein can help mitigate off-target effects and increase the safety profile,” Zhang explained. “This is because off-targets tend to occur when there is an excess of CRISPR reagents or when they linger in the cells for too long. Applying inhibitors to restrict the amount or duration of CRISPR action has proven to be effective in reducing off-target edits while maintaining on-target edits.”

The team aimed to develop a reliable off switch for the Cas3 system, previously identified in the harmless Neisseria lactima bacteria. By screening anti-CRISPRs reported in the literature as inhibitors for various Cas3 variants, they identified two, AcrIC8 and AcrIC9, with potent cross-reactive effects against the Neisseria Cas3.

Delving into the mechanics of these anti-CRISPRs, Zhang noted that “how they work will inform what kind of Cas3-based technologies can be controlled by each anti-CRISPR.”

Through genetic and biochemical studies at U-M and cryogenic electron microscopy at Cornell, the team unraveled the action mechanism and molecular structure of AcrlC8 and AcrlC9. Both proteins obstruct the CRISPR-Cas3 complex's ability to bind to its DNA target, though via slightly different methods.

Zhang expressed astonishment at the atomic-level understanding achieved by Ke lab's work, highlighting the proteins’ strategy of competing with the DNA for Cas machinery binding, effectively blocking Cas protein function.

Importantly, the research provided key evidence that these anti-CRISPR proteins can serve as off-switches for CRISPR-Cas3 in human cells, establishing them as the first off-switches for any CRISPR-Cas3 gene editor.

“Cas3 with an off switch would be a safer way to engineer the genome,” Zhang stated, indicating her lab's intention to continue developing CRISPR-Cas3-based therapeutics for various human diseases at U-M Medical School with these newly discovered off switches.

Chunyi Hu, Ph.D. of Cornell University, and Ph.D. student Mason Myers of U-M are the first authors of the manuscript, reflecting Myers' significant developmental trajectory as a young scientist under Zhang's guidance.