The Knock-In Atlas: A web resource for targeted protein trap by CRISPR/Cas9 in human and mouse cell lines

Various cell engineering techniques have been developed by leveraging the CRISPR-Cas9 technology, but large-scale resources for targeted gene knock-in are still limited. Here we introduce a tool kit for tagging genes by inserting artificial exons encoding fluorescent protein tags in target gene introns. To produce knock-in cells efficiently and reproducibly, we carefully chose and catalogued guide RNAs (gRNAs) for targeting genes in the human and mouse genomes by taking the gRNA efficacy scores and protein structures around the insertion sites into account. So far, we have constructed 427 gRNA expression plasmids to target 178 genes as the first set. The transfection and flow cytometry protocols were optimized for several cell lines including HEK293T, eHAP1, HeLa, THP-1, Neuro2a, mouse embryonic fibroblast (MEF) and mouse embryonic stem cell (mESC). A website has been launched to organize the results of initial characterization including flow cytometry data after transfection, confocal microscopy, and western blot results for the genes for which knock-in HEK293T cell lines were already made. We provide a user-friendly database to organize the information of the cell line and pre-designed gRNAs at <https://yumahanaiatokamuralab.shinyapps.io/KnockInAtlas/>.