Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing

In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.¹.