Development of a method for revertable CRISPR/Cas9-based mutagenesis in cell culture

Walsh, J.; Eggenschwiler, J.

doi:10.1101/2020.09.07.286187

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bioRxiv · 10.1101/2020.09.07.286187

Development of a method for revertable CRISPR/Cas9-based mutagenesis in cell culture

Walsh, J.·Eggenschwiler, J.

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Abstract

CRISPR/Cas9 mutagenesis is a revolutionary tool for genetics in organismal and cell culture systems. One notable caveat with this system is the potential for phenotype-inducing off-target/background mutations. There has been considerable success in modifying the methodology to minimize these potential confounds. Here we have developed a tool to functionally demonstrate that a targeted mutation of interest is responsible for the phenotype observed. This approach creates revertable mutations in cell culture systems using CRISPR/Cas9-induced homology-directed repair (HDR) to insert a LoxP-flanked transcriptional stop sequence into an early intron of a target gene. This method has the potential to be used in multiplexed and inducible scenarios to restore gene function within a given experiment.

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Walsh, J., Eggenschwiler, J.. 2020-09-07. Development of a method for revertable CRISPR/Cas9-based mutagenesis in cell culture. https://doi.org/10.1101/2020.09.07.286187

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