CRISPR/Cas9-mediated Base-editing Enables a Chain Reaction Through Sequential Repair of sgRNA Scaffold Mutations

Abstract

Cellular behavior is governed by the complex gene regulatory networks. Although studies have revealed diverse roles of individual genes, it has been a challenge to record or control the sequential genetic events in living cells. In this study, we designed two cellular chain reaction systems that enable sequential sgRNA expression in mammalian cells using a nickase Cas9 tethering of a cytosine nucleotide deaminase (nCas9-CDA). In these systems, the thymidine (T)-to-cytosine (C) substitutions in the scaffold region of sgRNA or TATA box containing loxP sequence (TATAloxP) are corrected by the nCas9-CDA, which leads to expression of next sgRNA. These reactions can proceed several times, thus generating cellular chain reactions. As a proof of the concept, we established a chain reaction through the repair of sgRNA scaffold mutations in 293T cells. Importantly, the results obtained in yeast or in vitro were not consistent with those in mammalian cells, suggesting that the in vivo chain reactions need to be optimized in appropriate cellular contexts. Our system may lay the foundation for building cellular chain reaction systems that have a broad utility in the future biomedical research.