Scientists have harnessed the revolutionary CRISPR technology, originally an immune system bacteria use to defend against viruses, to edit genetic information within cells. The FDA recently approved the first CRISPR-based therapeutic in December 2023, specifically designed to treat sickle cell disease using the well-studied CRISPR-Cas9 genetic scissor system.

While CRISPR-Cas9 has paved the way for gene editing, a newer and promising platform, Type I CRISPR or CRISPR-Cas3, with the potential for large-sized DNA removals, is emerging for potential therapeutic use.

A recent study led by Yan Zhang, Ph.D., Assistant Professor in the Department of Biological Chemistry at the University of Michigan Medical School, in collaboration with Cornell University, focuses on improving the safety of the Type I-C/Cas3 gene editor. Published in the journal Molecular Cell, the study titled “Exploiting Activation and Inactivation Mechanisms in Type I-C CRISPR-Cas3 for Genome Editing Applications” unveils novel off-switches designed to enhance safety.

The researchers drew inspiration from the ongoing arms race between bacteria and viruses (bacteriophages) to develop CRISPR off-switches. These switches, derived from anti-CRISPR proteins evolved by phages to counter bacterial CRISPR immunity, aim to address off-target effects associated with CRISPR-Cas9.

Zhang emphasized the importance of using inhibitor proteins to mitigate off-target effects and increase the safety profile of CRISPR-Cas9. The team then set out to create a robust off switch for the highly efficient Cas3 system, identified in bacteria residing in the human upper respiratory tract.

Screening known anti-CRISPRs for other Cas3 variants, the team identified AcrIC8 and AcrIC9 with potent cross-reacting effects against Neisseria Cas3. Through genetic and biochemical studies at the University of Michigan and cryogenic electron microscopy analysis at Cornell, they unveiled the molecular-level mechanisms of action and structure of these anti-CRISPR proteins.

The team’s key proof-of-concept findings demonstrate that AcrIC8 and AcrIC9 can serve as effective off-switches for CRISPR-Cas3 in human cells. These off-switches can almost completely block two versions of CRISPR-Cas3 technologies, making them the first off-switches developed for any CRISPR-Cas3 gene editor.

Zhang envisions a safer way to engineer the genome using Cas3 with an off switch. Her lab plans to further develop CRISPR-Cas3-based therapeutics for various human diseases, collaborating with colleagues at the University of Michigan Medical School. The study’s first authors are Chunyi Hu, Ph.D., from Cornell University, and Ph.D. student Mason Myers from the University of Michigan. Co-corresponding author Ailong Ke, Ph.D., from Cornell University, highlights the potential of these findings in advancing the field of genome editing.

By Impact Lab