CRISPR-Assisted RNA–Protein Interaction Detection

Background

RNA–protein interactions govern virtually every aspect of cellular metabolism, including gene expression, RNA processing, translation, and ribonucleoprotein assembly. With the discovery of vast networks of noncoding RNAs and thousands of putative RNA-binding proteins, understanding how these molecules physically associate has become a primary focus in functional genomics and structural biology. Because disruptions in these complex regulatory networks are frequently implicated in human diseases, there is a critical need for robust, scalable molecular platforms capable of accurately detecting, mapping, and analyzing the specific binding determinants that drive ribonucleoprotein complex formation in living cells.

However, characterizing these interactions using existing methodologies remains a significant technical bottleneck. Traditional biochemical approaches rely on laborious, low-throughput in vitro purification steps that often disrupt native cellular contexts and fail to capture dynamic assemblies. Conversely, current genetic screening assays, such as the yeast three-hybrid system, frequently lack the sensitivity required to detect weak, transient, or low-abundance interactions. These conventional in vivo platforms also struggle to accommodate large, structurally complex RNA transcripts due to transcriptional and folding constraints, limiting the ability of researchers to perform high-resolution mutational mapping or conduct unbiased library screens against intricate biological targets.

Technology

The CRISPR-assisted RNA•RBP yeast (CARRY) two-hybrid system is an in vivo molecular screening platform designed to detect, map, and analyze RNA–protein interactions. Adapting the classical yeast two-hybrid paradigm, the system replaces protein-based DNA-binding baits with a catalytically inactive Cas9 (dCas9) ribonucleoprotein architecture. Target RNAs are fused to a CRISPR single-guide RNA (sgRNA) via a flexible multiple cloning site and flanked by self-cleaving ribozymes to ensure uniform processing. Co-expressed dCas9 binds this hybrid sgRNA, tethering it to specific operator sequences upstream of reporter genes like HIS3 and LacZ. Candidate RNA-binding proteins (RBPs) are fused to a transcriptional activation domain; when an RBP binds the target RNA, it activates reporter transcription, enabling visual or growth-based detection.

This technology is differentiated by its ability to capture weak, transient, or low-abundance RNA–protein interactions, achieving a 100- to 1000-fold sensitivity increase over standard configurations. This heightened sensitivity is driven by using potent endogenous promoters (TDH3 or YDR524C-B) to maximize fusion protein expression without cellular toxicity, alongside high-copy episomal reporter plasmids, robust alternative activation domains, and gain-of-function dCas9 mutations. Consequently, CARRY enables high-resolution mutational scanning and the pruning of bulky ribonucleoproteins—such as reducing telomerase RNA from 1,157 to a 152-nucleotide core—facilitating structural biology and unbiased library screening.

Benefits

• Enables in vivo detection, mapping, and analysis of RNA-protein interactions
• Achieves a 100- to 1000-fold increase in sensitivity to detect weak, transient, or low-abundance interactions
• Preserves colony viability and avoids cellular toxicity despite high intracellular fusion protein expression
• Facilitates seamless cloning and uniform post-transcriptional processing of diverse target RNA sequences and structural domains
• Supports high-resolution mutational and deletion mapping to identify essential binding motifs and domains
• Enables rational pruning of bulky ribonucleoproteins into compact, functionally active minimal cores for structural biology applications
• Provides a scalable, genetically encoded workflow for unbiased screening of protein libraries against disease-associated noncoding RNAs

Applications

• RNA-protein interaction screening
• Drug target discovery
• Biomarker identification
• Structural biology construct optimization
• Ribonucleoprotein binding domain mapping

Additional info

PCT Application: WO2018200653A1

Original Assignee: Johns Hopkins University

Inventors: David Zappulla and Evan P. Hass

Publication Date: November 1, 2018 Google Patents