CRISPR-Cas9 is revolutionizing the life sciences through its RNA-guided ability to target individual genes for disruption or for precise editing. In addition to accelerating basic research and biotechnology, Cas9 and its RNA guides also have the potential to transform the treatment of inherited diseases via genome editing-based cures, especially if delivery hurdles can be overcome and clinical safety can be proven. There are three subtypes of Cas9 systems (Types II-A, -B and -C), and all three have yielded Cas9 orthologs with demonstrated utility in mammalian genome editing. Our own work has established the Type II-C Cas9 from N. meningitidis (NmeCas9) as a compact, naturally hyperaccurate genome editing platform. Some of the safety concerns in Cas9?s clinical development arise from Cas9 activity that is excessive, prolonged, or present in unintended cell types. These concerns have been heightened by a shortage of effective tools that inactivate Cas9 proteins after completion of the intended editing, or that prevent Cas9 activity altogether at undesired times or in unintended tissues. We recently discovered natural Cas9 inhibitors [anti-CRISPR (Acr) proteins] that evolved as counter- measures against CRISPR immunity. We have identified and validated five distinct families of Acrs from bacterial species with Type II-C CRISPR-Cas systems, and established their efficacy as off-switches for NmeCas9 genome editing in human cells. Our proven strategies have also led us to additional Acr candidates that are likely inhibitors of distinct Type II-C Cas9 orthologs. Our discoveries therefore provide us with a powerful entre into addressing the limitations and safety concerns that arise from uncontrolled or otherwise unwanted Cas9 activity. In this proposal, we outline experiments that exploit our discovery of Cas9 inhibitors in three ways.
In Aim 1, we will use Acr phylogenetic distributions as pointers towards orthologous, robust, compact Type II-C Cas9 genome-editing systems, each of which would be immediately amenable to off-switch control. Because distinct Cas9s often have novel targeting specificities, this work also promises to expand the genomic scope of Cas9 editing.
In Aim 2, we will optimize Acr inhibitory potency, and test the hypothesis that Acrs can be used to increase the efficiency of precise editing via homology-dependent repair. Finally, in Aim 3, we will establish tissue-specific Acr control over genome editing in a model mammal (the mouse), and use it to develop a flexible platform for restricting Cas9 genome editing to a single desired tissue following systemic Cas9 delivery by adeno-associated virus. Our strategy for enforcing tissue specificity of gene editing will be applicable even to cell types in which AAV-compatible tissue-specific promoters are unavailable for driving Cas9 expression. The proposed research promises to yield enhanced genome-editing systems with broader targeting range, fewer safety risks and improved tissue specificity, which we will demonstrate pre-clinically in mice.

Public Health Relevance

The proposed research focuses on the development of ?off-switch? control for Cas9 genome editing. We recently discovered the first natural inhibitors of Cas9 bacterial immune systems, and we showed that they have unique potential for enabling control over the duration, extent, and tissue specificity of this revolutionary genome editing technology. Our work will use this off-switch control to improve the safety and efficacy of genome editing in its clinical development for the treatment of inherited disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM125797-01
Application #
9427037
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Willis, Kristine Amalee
Project Start
2018-02-13
Project End
2022-01-31
Budget Start
2018-02-13
Budget End
2019-01-31
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655