Protocol for high-throughput screening of functional lysine residues in cell fitness

Amino acid residues are crucial to protein structure and function and have links to various human diseases. Here, we present a protocol for screening functional lysine residues across the human genome. We describe steps for designing lysine codon-targeting single-guide RNAs (sgRNAs), constructing an sgRNA library, conducting cell fitness screenings, and acquiring screening results. This approach leverages base editing and high-throughput screening techniques to systematically examine functional amino acid residues. For complete details on the use and execution of this protocol, please refer to Bao et al.1 [Display omitted] •Steps for designing a lysine codon-targeting sgRNA library across the human genome•Guidance on constructing the library using iBAR technology at high MOI•Instructions for the execution of cell fitness screening in RPE1 cells Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Amino acid residues are crucial to protein structure and function and have links to various human diseases. Here, we present a protocol for screening functional lysine residues across the human genome. We describe steps for designing lysine codon-targeting single-guide RNAs (sgRNAs), constructing an sgRNA library, conducting cell fitness screenings, and acquiring screening results. This approach leverages base editing and high-throughput screening techniques to systematically examine functional amino acid residues.