A universal dark-to-bright GFP reporter for gene editing

Abstract

Current fluorescent reporters for assessing activity of CRISPR-Cas system, such as Stoplight or split-GFP based on frameshift activation, often suffer from limitations including large vector size, low viral packaging efficiency, and insensitivity to in-frame insertions or deletions. Although specialized reporters for base editing (e.g., BEAR) and prime editing (e.g., PEAR and fluoPEER) have been developed, there remains a significant demand for a compact, sensitive, and universal single-fluorescent reporter compatible with diverse editing types.

We constructed a novel reporter system based on a single fluorescent protein employing a "dark-state quenching to bright-state activation" strategy, named CRISPR-Bright (CRB). Its core design involves fusing a green fluorescent protein with a 28-amino-acid quenching peptide derived from the influenza M2 protein. In unedited station, the fusion protein forms tetramers mediated by the quenching peptide, completely suppressing GFP fluorescence. When Cas9 or prime editor generates insertions or deletions (±1 or ±2 nt frameshift) or a designed premature stop codon at the target site, the coding sequence of the quenching peptide is disrupted or terminated. Upon impaired expression of the quenching peptide, GFP folds, matures, and emits strong green fluorescence. We engineered this design into a lentiviral vector with a total size of only 5.8 kb, significantly improving viral packaging and delivery efficiency.

The result showed the CRB reporter efficiently reports Cas9 and prime editor mediated Indel events and enables sensitive, visual monitoring of prime editor activity. Its performance is notably superior to existing dual-fluorescent or large-vector-based reporter systems.