Programmable Transcriptional Modulation with a Structured RNA-Mediated CRISPR-dCas9 Complex

Abstract

Multi-module dCas9 engineering systems have been developed for controllable transcriptional manipulation such as chemical-or light-induced systems. However, there is still a need for a separate module that can be used for internal control over the CRISPR-dCas9 system. Here, we describe a multi-module CRISPR-dCas9 system in which a separate structured RNA was applied as a programmable component that could control dCas9-based gene regulation and achieved a higher activation efficiency than dCas9-VPR that is traditionally used. By introducing a microRNA sensor, we generated a dCas9-based transcriptional regulation platform that responded to endogenous microRNAs and allowed controllable activation of endogenous genes. Moreover, we applied the platform to selectively identify HCT116 cells in a cell mixture. This work provides a flexible platform for efficient and controllable gene regulation based on CRISPR-dCas9.

Stalling of Transcription by Putative G-quadruplex Sequences and CRISPR-dCas9

“…The CRISPR-dCas9 mediated transcription activation (CRISPRa) is straightforward, target-specific, programmable, and frequently applicable when compared to conventional expression techniques that are based on the modification of genes and promoters (5, 9). It also enables the recruitment of various effector proteins for gene regulation at the transcriptional level (10, 11). In E. coli , CRISPR interference (CRISPRi) technology was used to show the effectiveness of dCas9 for sequence-specific gene repression.…”

Unexploited Performance of NLS in the dCas9-VPR-Mediated Transcriptional Activation

“…4 In particular, the NLS has been ingeniously implemented in the clustered regularly interspaced short palindromic repeats (CRISPR) systems for genome editing 5,6 and gene regulation. [7][8][9][10][11][12]24 CRISPR-Cas proteins are an important class of nucleic acid tool enzymes. There were numerous reports about the effects of the NLS on Cas-related gene editing.…”

“…In recent years, the NLS has been widely applied in cell imaging and the cellular sublocalization of functional proteins . In particular, the NLS has been ingeniously implemented in the clustered regularly interspaced short palindromic repeats (CRISPR) systems for genome editing , and gene regulation. − , …”