Abstract
CRISPR system is a powerful gene editing tool which has already been reported to address a variety of gene relevant diseases in different cell lines. However, off-target effect and immune response caused by Cas9 remain two fundamental problems. Inspired by previously reported Cas9 self-elimination systems, time-delayed safety switches are designed in this work. Firstly, ultrasensitive relationship is constructed between Cas9-sgRNA (enzyme) and Cas9 plasmids (substrate), which generates the artificial time delay. Then intrinsic time delay in biomolecular activities is revealed by data fitting and utilized in constructing safety switches. The time-delayed safety switches function by separating the gene editing process and self-elimination process, and the tunable delay time may ensure a good balance between gene editing efficiency and side effect minimization. By addressing gene therapy efficiency, off-target effect, immune response and drug accumulation, we hope our safety switches may offer inspiration in realizing safe and efficient gene therapy in humans.
Highlights
CRISPR system is a powerful gene editing tool which has already been reported to address a variety of gene relevant diseases in different cell lines
CRISPR system consists of a DNA endonuclease enzyme Cas[9] and a single guide RNA2. sgRNA will bind with Cas[9], recognize target DNA and localize Cas[9] on the target
We proved that the time-delayed safety switch is superior to the linear pattern safety switch in one gene therapy case, and we demonstrate that the length of delay time is highly tunable for different gene therapy cases based on mathematical modeling results
Summary
Inspired by previously reported Cas[9] self-elimination systems, time-delayed safety switches are designed in this work. By addressing gene therapy efficiency, off-target effect, immune response and drug accumulation, we hope our safety switches may offer inspiration in realizing safe and efficient gene therapy in humans. We may expect the Cas[9] to remain a certain amount for a high gene therapy efficiency; after gene therapy is complete, Cas[9] should be eliminated as fast as possible to minimize side effects. Featured by a time-delayed design, our safety switches have the potential to guarantee gene therapy efficiency when minimizing both off-target and immune response risk, thereby enable safe and efficient gene therapy in humans
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