A strategy for genome-wide seamless tagging of human protein-coding genes

Abstract

Comprehensive and systematic proteome-wide experiments require financial and technical resources unavailable to most researchers. Here we describe a universal and scalable method based on CRISPR/Cas9-non-homologous end joining (NHEJ) for Pooled Recombinant Integration of Seamless Markers (PRISM) into protein coding genes. We created two genome- wide gRNA libraries for 5’- and 3’-tagging of 18,489 human protein-coding genes. Selection for in-frame integration of the donor cassette can be favored by fusing it to an antibiotic resistance enzyme (ARE) and P2A self-cleaving peptide, resulting in tagged proteins and free ARE to select for clones with inserted donor cassettes. We achieved a library integration rate of ∼20% and tagging of at least 36% of genes expressed in HEK 293T cells including, at least 28% of essential genes, with donor DNA. Our strategy is universal, scalable, specific, and selective, paving the way for genome-scale construction of human cell lines tagged with different types of reporter genes for protein functional characterization. Significance Statement Here we report a practical method to achieve 5’- or 3’-end integration of reporter protein-coding sequences to express seamless fusions of human protein and reporter proteins that we call Pooled Recombinant Integration of Seamless Markers (PRISM). PRISM is independent of homology templates, yet it works with high efficiency and precision. Using gRNA-directed CRISPR/Cas9 genome editing and mini-plasmid donor cassettes, we were able to tag at least 36% of the protein- coding sequences of human genes with a green fluorescent protein at an integration efficiency of about 20% in the human cell line HEK 293T. This included tagging approximately 28% of essential genes, an important fact considering their relevance for gene characterization in different cell types.