Krishanu Saha, PhD – Slide of the Week

Krishanu Saha, PhD - Slide of the Week

Title: Controlling CRISPR with small molecule regulation for somatic cell genome editing

Legend: Three translational challenges for in vivo somatic cell genome editing with CRISPR-Cas9. (1) Off-target DNA double-strand break formation. The gene of interest is targeted by the single guide RNA (sgRNA) and cut by Cas9, and it results in double-stranded breaks that can subsequently be repaired to generate on-target edits. However, with prolonged exposure of Cas9 to the genome, there can be an increase in unwanted modifications at off-target sites. (2) Antibody neutralization to delivery vectors. Antigen-presenting cells may bind capsid proteins or other components from the delivery vector (e.g., polyethylene glycol [PEG]) to trigger an antibody-mediated immune response. (3) Immune response to Cas and vector proteins. MHC class I molecules may bind peptides from degradation of Cas9 and/or the viral vector and present them on the cell surface. These peptides could be presented to T cells and trigger an adaptive immune response.

Citation: Khajanchi, N., & Saha, K. (2022). Controlling CRISPR with small molecule regulation for somatic cell genome editing. Molecular therapy : the journal of the American Society of Gene Therapy, 30(1), 17–31.

Abstract: Biomedical research has been revolutionized by the introduction of many CRISPR-Cas systems that induce programmable edits to nearly any gene in the human genome. Nuclease-based CRISPR-Cas editors can produce on-target genomic changes but can also generate unwanted genotoxicity and adverse events, in part by cleaving non-targeted sites in the genome. Additional translational challenges for in vivo somatic cell editing include limited packaging capacity of viral vectors and host immune responses. Altogether, these challenges motivate recent efforts to control the expression and activity of different Cas systems in vivo. Current strategies utilize small molecules, light, magnetism, and temperature to conditionally control Cas systems through various activation, inhibition, or degradation mechanisms. This review focuses on small molecules that can be incorporated as regulatory switches to control Cas genome editors. Additional development of CRISPR-Cas-based therapeutic approaches with small molecule regulation have high potential to increase editing efficiency with less adverse effects for somatic cell genome editing strategies in vivo.

About the Lab: The Saha Lab (Kris Saha) is affiliated with several multi-disciplinary centers including the Waisman Center, Wisconsin Institute for Discovery and the Stem Cell and Regenerative Medicine Center at UW-Madison. Our research dedicated to using human stem cells together with emerging engineering methods in material science and synthetic biology to make smarter therapeutics, model human disease, and advance personalized medicine. We are collaborative efforts on campus in biomanufacturing cell and gene therapies, and nationally with the NIH Somatic Cell Genome Editing program and the NSF Center for Cell Manufacturing Technologies.

Investigator: Krishanu Saha, PhD

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