Work

SELECT: high-precision genome editing strategy via integration of CRISPR–Cas and DNA damage response for cross-species applications

Year: 2025

Type: article

Abstract: CRISPR-based methods enable genome modifications for diverse applications but often face challenges, such as inconsistent efficiencies, reduced performance in iterative modifications, and difficulties generating high-quality datasets for high-throughput genome engineering. Here, we present SELECT (SOS Enhanced programmabLE CRISPR-Cas ediTing), a novel strategy integrating the CRISPR-Cas system with the DNA damage response. By employing designed and optimized double-strand break induced promoters that are activated upon genome editing, SELECT enables a counter-selection process to eliminate unedited cells, ensuring high-fidelity editing. This approach achieves up to 100% efficiency for point mutations, iterative knockouts, and insertions. In high-throughput library editing, SELECT achieved up to 94.2% efficiency and preserved higher library diversity compared with conventional methods. Application of SELECT in flaviolin biosynthesis resulted in a 3.97-fold increase in production. Furthermore, integration with machine learning tools allowed rapid mapping of genotype-phenotype relationships. SELECT provides a versatile platform for precision genome engineering in Escherichia coli and Saccharomyces cerevisiae.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gkaf595