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Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells
Chase P Kelley1, Maja C Haerle2, Eric T Wang3
1Department of Molecular Genetics & Microbiology, Center for NeuroGenetics, Genetics Institute, University of Florida, Gainesville, FL 32608, USA; Genetics and Genomics Graduate Program, University of Florida, Gainesville, FL 32608, USA.
Abstract:
CRISPR-Cas13 RNA endonucleases show promise for programmable RNA knockdown. However, sequence-specific binding of Cas13 unleashes non-specific bystander RNA cleavage, or collateral activity, raising concerns for experiments and therapeutic applications. Although robust in cell-free and bacterial environments, collateral activity in mammalian cells remains disputed. We investigate Cas13d collateral activity in a therapeutic context for myotonic dystrophy type 1, caused by a transcribed CTG repeat expansion. We find that, when targeting CUGn RNA in mammalian cells, Cas13d depletes endogenous and transgenic RNAs, interferes with critical cellular processes, and activates stress response and apoptosis. Collateral effects also occur when targeting abundant endogenous transcripts. To minimize collateral activity for repeat-targeting approaches, we introduce GENO, an adeno-associated virus-compatible strategy that leverages guide RNA processing to control Cas13d expression. We argue that thorough assessment of collateral activity is necessary when applying Cas13 in mammalian cells and that GENO illustrates advantages of compact regulatory systems for Cas-based gene therapies.
Insights
CRISPR-Cas13d RNA knockdown in mammalian cells causes unintended RNA cleavage and cellular damage. A new strategy, GENO, controls Cas13d expression to minimize these harmful collateral effects for gene therapy.
Area of Science:
- Molecular Biology
- Gene Therapy
- Biochemistry
Background:
- CRISPR-Cas13 RNA endonucleases offer programmable RNA knockdown capabilities.
- Cas13's sequence-specific binding can trigger non-specific RNA cleavage (collateral activity), a concern in therapeutic applications.
- Collateral activity of Cas13 in mammalian cells is not fully understood.
Purpose of the Study:
- To investigate Cas13d collateral activity in mammalian cells within a therapeutic context for myotonic dystrophy type 1.
- To develop a strategy to minimize Cas13d collateral activity for RNA-targeting approaches.
Main Methods:
- Utilized CRISPR-Cas13d to target CUG repeat RNA in mammalian cells.
- Assessed RNA depletion, cellular process interference, and stress/apoptosis responses.
- Introduced GENO, an adeno-associated virus-compatible strategy using guide RNA processing to control Cas13d expression.
Main Results:
- Cas13d targeting CUG repeat RNA depleted endogenous and transgenic RNAs, interfered with cellular processes, and induced stress and apoptosis.
- Collateral effects were observed even when targeting abundant endogenous transcripts.
- The GENO strategy effectively controlled Cas13d expression and minimized collateral activity.
Conclusions:
- Thorough assessment of Cas13 collateral activity is crucial for mammalian cell applications.
- The GENO strategy demonstrates the potential of compact regulatory systems for developing safer Cas-based gene therapies.
- Minimizing collateral effects is essential for the safe and effective use of CRISPR-Cas13 in therapeutics.
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