{"doi":"10.1002/cpz1.70041","title":"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.","journal":"Current Protocols","year":2024,"id":477940,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9463,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":99042,"name":"Benjamin E. Gewurz","orcid":"0000-0002-3965-3418","position":1,"is_corresponding":false},{"id":277732,"name":"Laura A. Murray‐Nerger","orcid":"0000-0001-7910-6235","position":0,"is_corresponding":true}],"reference_count":11,"raw_metadata":null,"created_at":"2026-07-19T02:06:42.155024Z","pmid":"39535522","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}