CRISPR signal conductor 2.0 for redirecting cellular information flow

Yonghao Zhan,Yuchen Liu,Aolin Li,Congcong Cao

doi:10.1038/s41421-021-00371-1

Abstract

A key challenge in designing intelligent artificial gene circuits is generating flexible connections between arbitrary components and directly coupling them with endogenous signaling pathways. The CRISPR signal conductor based on conditionally inducible artificial transcriptional regulators can link classic cellular protein signals with targeted gene expression, but there are still problems with multiple signal processing and gene delivery. With the discovery and characterization of new Cas systems and long noncoding RNA (lncRNA) functional motifs, and because of the compatibility of guide RNA with noncoding RNA elements at multiple sites, it is increasingly possible to solve these problems. In this study, we developed CRISPR signal conductor version 2.0 by integrating various lncRNA functional motifs into different parts of the crRNA in the CRISPR-dCasΦ system. This system can directly regulate the expression of target genes by recruiting cellular endogenous transcription factors and efficiently sense a variety of protein signals that are not detected by a classical synthetic system. The new system solved the problems of background leakage and insensitive signaling responses and enabled the construction of logic gates with as many as six input signals, which can be used to specifically target cancer cells. By rewiring endogenous signaling networks, we further demonstrated the effectiveness and biosafety of this system for in vivo cancer gene therapy.

Highlights

To solve the problems of previous clustered regularly interspaced short palindromic repeat (CRISPR) signal conductors, we developed the CRISPR signal conductor version 2.0 by integrating various long noncoding RNA (lncRNA) functional motifs into different parts of the CRISPR RNA in deactivated CasΦ variant (dCasΦ)
LncRNAs were functionally appended onto the single guide RNA (sgRNA) of Cas[9] at multiple positions, including the 5′-end, 3′-end, and middle stem-loop region of the sgRNA38
We analyzed the composition of guide RNA (gRNA) of the CRISPR-CasΦ-2 system, which consisted of a 44-nt CRISPR RNA (crRNA) with a short hairpin structure for binding CasΦ protein and a target-specific antisense RNA fused to the 3′-end of the crRNA

Summary

Introduction

Cells respond to diverse biological signals through natural gene circuits[1], and timely feedback improves their ability to adapt to complex external environments. Intelligent artificial gene circuits have been established, composability remains a challenge, Zhan et al Cell Discovery (2022)8:26 the ability to connect arbitrary components and directly couple them with endogenous signaling pathways. To solve this problem, two main strategies have been developed; in one solution, protein–protein interactions are engineered[14,15], and in the other solution, RNA-based riboregulators are designed[16,17]. Combining protein–protein regulatory systems can produce a variety of circuit structures, which facilitate rational gene circuit design, and through their interactions, proteins can respond to signals very quickly[18]. RNAs can be unstable and degraded, and it may be necessary to build a library to screen RNAs with the best regulatory effects on target genes[19]

Methods

Results

Conclusion