{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T03:16:51Z","timestamp":1772767011372,"version":"3.50.1"},"reference-count":18,"publisher":"Wiley","issue":"11","license":[{"start":{"date-parts":[[2024,11,13]],"date-time":"2024-11-13T00:00:00Z","timestamp":1731456000000},"content-version":"vor","delay-in-days":12,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2024,11]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Precise understanding of temporally controlled protein\u2010protein interactions, localization, and expression is often difficult to achieve using traditional overexpression techniques. Recent advances have made CRISPR\u2010based knock\u2010in approaches efficient, which enables rapid derivation of cells with tagged endogenous proteins. However, the high degree of variability in knock\u2010in 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\u2010mediated knock\u2010in. We address knock\u2010in 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\u2010expressing them with an unlinked fluorescent marker. Either approach facilitates downstream selection of single\u2010cell or bulk populations with the desired knock\u2010in, particularly when knock\u2010in efficiency is low. The utility of this approach is demonstrated via examples of engineering tags onto endogenous protein N\u2010 or C\u2010termini, together with downstream analyses. We anticipate that this workflow can be applied more broadly to other cell types for efficient knock\u2010in into endogenous loci. \u00a9 2024 Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Choosing an optimal knock\u2010in target site and single guide RNA (sgRNA) design<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Assessment of Cas9 editing efficiency at the desired B cell genomic knock\u2010in site<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Cloning the sgRNA dual guide construct<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Repair template design and cloning<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Electroporation and selection of engineered B cells<\/jats:p><jats:p><jats:bold>Basic Protocol 6<\/jats:bold>: Single\u2010cell cloning of engineered B cells<\/jats:p>","DOI":"10.1002\/cpz1.70041","type":"journal-article","created":{"date-parts":[[2024,11,13]],"date-time":"2024-11-13T13:29:36Z","timestamp":1731504576000},"source":"Crossref","is-referenced-by-count":2,"title":["Efficient CRISPR\/Cas9 Knock\u2010in Approaches for Manipulation of Endogenous Genes in Human B Lymphoma Cells"],"prefix":"10.1002","volume":"4","author":[{"given":"Laura A.","family":"Murray\u2010Nerger","sequence":"first","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine Brigham and Women's Hospital  Boston Massachusetts"},{"name":"Center for Integrated Solutions to Infectious Diseases Broad Institute of Harvard and MIT  Cambridge Massachusetts"},{"name":"Department of Microbiology Harvard Medical School  Boston Massachusetts"},{"name":"PhD Program in Virology Harvard University  Boston Massachusetts"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3965-3418","authenticated-orcid":false,"given":"Benjamin E.","family":"Gewurz","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine Brigham and Women's Hospital  Boston Massachusetts"},{"name":"Center for Integrated Solutions to Infectious Diseases Broad Institute of Harvard and MIT  Cambridge Massachusetts"},{"name":"Department of Microbiology Harvard Medical School  Boston Massachusetts"},{"name":"PhD Program in Virology Harvard University  Boston Massachusetts"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,11,13]]},"reference":[{"key":"e_1_2_11_2_1_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4265"},{"key":"e_1_2_11_2_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chembiol.2020.04.003"},{"key":"e_1_2_11_2_3_1","doi-asserted-by":"publisher","DOI":"10.1002\/cpmb.51"},{"key":"e_1_2_11_2_4_1","doi-asserted-by":"publisher","DOI":"10.1261\/rna"},{"key":"e_1_2_11_2_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3190"},{"key":"e_1_2_11_2_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mie.2022.08.045"},{"key":"e_1_2_11_2_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467\u2010024\u201048333\u2010x"},{"key":"e_1_2_11_2_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41589\u2010018\u20100021\u20108"},{"key":"e_1_2_11_2_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms6560"},{"key":"e_1_2_11_2_10_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv993"},{"key":"e_1_2_11_3_2_1","unstructured":"https:\/\/chopchop.cbu.uib.no\/"},{"key":"e_1_2_11_3_3_1","unstructured":"The CHOP\u2010CHOP site is a useful resource for sgRNA design and for design of the homologous repair construct."},{"key":"e_1_2_11_3_4_1","unstructured":"https:\/\/www.synthego.com\/products\/bioinformatics\/crispr\u2010analysis"},{"key":"e_1_2_11_3_5_1","unstructured":"The Synthego ICE online tool is a useful resource for determining the editing efficiency (% indel) specified by Cas9 together with an sgRNA that targets the desired genomic knock\u2010in site."},{"key":"e_1_2_11_3_6_1","unstructured":"https:\/\/web.expasy.org\/translate\/"},{"key":"e_1_2_11_3_7_1","unstructured":"We use the Expasy Translate tool to check the homologous repair template reading frame."},{"key":"e_1_2_11_3_8_1","unstructured":"https:\/\/www.thermofisher.com\/us\/en\/home\/life\u2010science\/cell\u2010culture\/transfection\/neon\u2010transfection\u2010system\/neon\u2010transfection\u2010system\u2010cell\u2010line\u2010data.html"},{"key":"e_1_2_11_3_9_1","unstructured":"The Neon Transfection System Cell Line Data and Transfection Parameters resource provided by Thermo Fisher Scientific is a useful starting point for optimization of electroporation parameters."}],"container-title":["Current Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpz1.70041","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,12,2]],"date-time":"2024-12-02T04:58:05Z","timestamp":1733115485000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpz1.70041"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,11]]},"references-count":18,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2024,11]]}},"alternative-id":["10.1002\/cpz1.70041"],"URL":"https:\/\/doi.org\/10.1002\/cpz1.70041","archive":["Portico"],"relation":{},"ISSN":["2691-1299","2691-1299"],"issn-type":[{"value":"2691-1299","type":"print"},{"value":"2691-1299","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,11]]},"article-number":"e70041"}}