Programmable mammalian translational modulators by CRISPR-associated proteins

Abstract

The complexity of synthetic genetic circuits relies on repertories of biological circuitry with high orthogonality. Although post-transcriptional circuitry relying on RNA-binding proteins (RBPs) qualifies as a repertory, the limited pool of regulatory devices hinders network modularity and scalability. Here we propose CaRTRIDGE (Cas-Responsive Translational Regulation Integratable into Diverse Genomic Engineering) to repurpose CRISPR-associated (Cas) proteins as translational modulators. We demonstrate that a set of Cas proteins are able to repress (OFF) or activate (ON) the translation of mRNAs that contain a Cas-binding RNA motif in the 5'-UTR. We designed 81 different types of translation OFF and ON switches and verified their functional characteristics. Many of them functioned as efficient translational regulators and showed orthogonality in mammalian cells. By interconnecting these switches, we designed and built artificial circuits, including 60 translational AND gates. Moreover, we show that various CRISPR-related technologies, including anti-CRISPR and split-Cas9 platforms, can be repurposed to control translation. Our Cas-mediated translational regulation is compatible with transcriptional regulation by Cas proteins and increases the complexity of synthetic circuits with fewer elements. CaRTRIDGE builds protein-responsive mRNA switches more than ever and leads to the development of both Cas-mediated genome editing and translational regulation technologies.

Customizing cellular signal processing by synthetic multi-level regulatory circuits

“…Besides RNA interactions, the RNA-binding proteins (RBPs) also exert control over numerous RNA processes. For example, proteins binding to RNA aptamers at eukaryotic mRNAs' UTR 68,69 and intron regions 70 modulate their translation and splicing activities. A remarkable work in expanding available sets of orthogonal protein-RNA interactions is the verification of 13 orthogonal Cas proteins with their cognate gRNA motifs 69 .…”

“…For example, proteins binding to RNA aptamers at eukaryotic mRNAs' UTR 68,69 and intron regions 70 modulate their translation and splicing activities. A remarkable work in expanding available sets of orthogonal protein-RNA interactions is the verification of 13 orthogonal Cas proteins with their cognate gRNA motifs 69 . Furthermore, RBPs participate in altering RNA turnover, mediated by antisense RNA (asRNA), microRNA (miRNA), and endoribonuclease (endoRNase).…”

“…The CRISPRibased NOT and NOR gates have been interconnected into two-input logic gates, among which the AND and NAND gates were built from six sgRNAs 109 . However, these gates can be constructed using fewer sgRNAs by incorporating crRNA-tracrRNA interaction with CRISPRa 110 or protein-splicing with CRISPRi systems 69 . The multipartite assembly of gRNAs and synthetic RNAs 111 further scaled up these computations.…”