Efficient CRISPR/Cas9 Knock‐in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells

Abstract Precise understanding of temporally controlled protein‐protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR‐based knock‐in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock‐in efficiency across cell types and gene loci poses challenges, in particular with B lymphocytes, which are refractory to lipid transfection. Here, we present detailed protocols for efficient B lymphoma cell CRISPR/Cas9‐mediated knock‐in. We address knock‐in efficiency in two ways. First, we provide a detailed approach for assessing cutting efficiency to select the most efficient single guide RNA for the gene region of interest. Second, we provide detailed approaches for tagging endogenous proteins with a fluorescent marker or instead for co‐expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single‐cell or bulk populations with the desired knock‐in, particularly when knock‐in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N‐ or C‐termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock‐in into endogenous loci. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : Choosing an optimal knock‐in target site and single guide RNA (sgRNA) design Basic Protocol 2 : Assessment of Cas9 editing efficiency at the desired B cell genomic knock‐in site Basic Protocol 3 : Cloning the sgRNA dual guide construct Basic Protocol 4 : Repair template design and cloning Basic Protocol 5 : Electroporation and selection of engineered B cells Basic Protocol 6 : Single‐cell cloning of engineered B cells