{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T22:24:45Z","timestamp":1790807085964,"version":"4.1.0"},"reference-count":45,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2018,1,16]],"date-time":"2018-01-16T00:00:00Z","timestamp":1516060800000},"content-version":"vor","delay-in-days":15,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CP Molecular Biology"],"published-print":{"date-parts":[[2018,1]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Epstein\u2010Barr virus (EBV) efficiently transforms primary human B cells into immortalized lymphoblastoid cell lines (LCLs), which are extensively used in human genetic, immunological and virological studies. LCLs provide unlimited sources of DNA for genetic investigation, but can be difficult to manipulate, for instance because low retroviral or lentiviral transduction frequencies hinder experiments that require co\u2010expression of multiple components. This unit details Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)\/Cas9 engineering for robust LCL genome editing. We describe the generation and delivery of single\u2010guide RNAs (sgRNAs), or dual\u2010targeting sgRNAs, via lentiviral transduction of LCLs that stably express Cas9 protein. CRISPR\/Cas9 editing allows LCL loss\u2010of\u2010function studies, including knock\u2010out of protein\u2010coding genes or deletion of DNA regulatory elements, and can be adapted for large\u2010scale screening approaches. Low transfection efficiencies are a second barrier to performing CRISPR editing in LCLs, which are not typically lipid\u2010transfectable. To circumvent this barrier, we provide an optimized protocol for LCL nucleofection of Cas9\/sgRNA ribonucleoprotein complexes (RNPs) as an alternative route to achieve genome editing in LCLs. These editing approaches can also be employed in other B\u2010cell lines, including Burkitt lymphoma and diffuse large B\u2010cell lymphoma cells, and are highly reproducible. \u00a9 2018 by John Wiley &amp; Sons, Inc.<\/jats:p>","DOI":"10.1002\/cpmb.51","type":"journal-article","created":{"date-parts":[[2018,1,16]],"date-time":"2018-01-16T18:13:46Z","timestamp":1516126426000},"source":"Crossref","is-referenced-by-count":35,"title":["CRISPR\/Cas9\u2010Mediated Genome Editing in Epstein\u2010Barr Virus\u2010Transformed Lymphoblastoid B\u2010Cell Lines"],"prefix":"10.1002","volume":"121","author":[{"given":"Sizun","family":"Jiang","sequence":"first","affiliation":[{"name":"Harvard Ph.D. Program in Virology, Division of Medical Sciences, Harvard University Boston Massachusetts"},{"name":"Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital Boston Massachusetts"},{"name":"Present Address: Baxter Laboratory for Stem Cell Biology, Stanford University School of Medicine Stanford California"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Liang Wei","family":"Wang","sequence":"additional","affiliation":[{"name":"Harvard Ph.D. Program in Virology, Division of Medical Sciences, Harvard University Boston Massachusetts"},{"name":"Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital Boston Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael J.","family":"Walsh","sequence":"additional","affiliation":[{"name":"Harvard Ph.D. Program in Virology, Division of Medical Sciences, Harvard University Boston Massachusetts"},{"name":"Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital Boston Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen J.","family":"Trudeau","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital Boston Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Catherine","family":"Gerdt","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital Boston Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bo","family":"Zhao","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital Boston Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Benjamin E.","family":"Gewurz","sequence":"additional","affiliation":[{"name":"Harvard Ph.D. Program in Virology, Division of Medical Sciences, Harvard University Boston Massachusetts"},{"name":"Division of Infectious Diseases, Department of Medicine, Brigham and Women's Hospital Boston Massachusetts"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2018,1,16]]},"reference":[{"key":"e_1_2_10_2_1_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmicrobiol.2017.92"},{"key":"e_1_2_10_2_2_1","unstructured":"Benchling[Biology Software]. (2017)."},{"key":"e_1_2_10_2_3_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gku936"},{"key":"e_1_2_10_2_4_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.A114.564625"},{"key":"e_1_2_10_2_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature15521"},{"key":"e_1_2_10_2_6_1","unstructured":"Retrieved fromhttps:\/\/www.nature.com\/nature\/journal\/v527\/n7577\/abs\/nature15521.html\u2010supplementary\u2010information."},{"key":"e_1_2_10_2_7_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1000287"},{"key":"e_1_2_10_2_8_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1231143"},{"key":"e_1_2_10_2_9_1","doi-asserted-by":"crossref","first-page":"3J:A.3J.1","DOI":"10.1002\/0471142727.mba03js93","article-title":"Microvolume Quantitation of Nucleic Acids","volume":"93","author":"Desjardins P.","year":"2011","journal-title":"Current Protocols in Molecular Biology"},{"key":"e_1_2_10_2_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.4264"},{"key":"e_1_2_10_2_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3437"},{"key":"e_1_2_10_2_12_1","unstructured":"Retrieved fromhttps:\/\/www.nature.com\/nbt\/journal\/v34\/n2\/abs\/nbt.3437.html\u2010supplementary\u2010information."},{"key":"e_1_2_10_2_13_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3026"},{"key":"e_1_2_10_2_14_1","doi-asserted-by":"crossref","first-page":"3D:A.3D.1","DOI":"10.1002\/0471142727.mba03ds93","article-title":"Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy","volume":"93","author":"Gallagher S. R.","year":"2011","journal-title":"Current Protocols in Molecular Biology"},{"key":"e_1_2_10_2_15_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004890"},{"key":"e_1_2_10_2_16_1","doi-asserted-by":"publisher","DOI":"10.1098\/rstb.2015.0496"},{"key":"e_1_2_10_2_17_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2014.05.010"},{"key":"e_1_2_10_2_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature02168"},{"key":"e_1_2_10_2_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2014.12.013"},{"key":"e_1_2_10_2_20_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1225829"},{"key":"e_1_2_10_2_21_1","first-page":"2603","volume-title":"Fields Virology","author":"Kieff E.","year":"2007"},{"key":"e_1_2_10_2_22_1","doi-asserted-by":"publisher","DOI":"10.1101\/gr.171322.113"},{"key":"e_1_2_10_2_23_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3450"},{"key":"e_1_2_10_2_24_1","doi-asserted-by":"publisher","DOI":"10.1101\/gr.136184.111"},{"key":"e_1_2_10_2_25_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2017.04.005"},{"key":"e_1_2_10_2_26_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1232033"},{"key":"e_1_2_10_2_27_1","doi-asserted-by":"publisher","DOI":"10.1002\/0471142727.mb1008s114"},{"key":"e_1_2_10_2_28_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1004822"},{"key":"e_1_2_10_2_29_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0140\u20106736(73)93286\u20108"},{"key":"e_1_2_10_2_30_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro.2017.29"},{"key":"e_1_2_10_2_31_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.2842"},{"key":"e_1_2_10_2_32_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1512503112"},{"key":"e_1_2_10_2_33_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature15393"},{"key":"e_1_2_10_2_34_1","first-page":"28.4:28.4.1","article-title":"Detection of Mycoplasma Contamination in Cell Cultures","volume":"106","author":"Uphoff C. C.","year":"2014","journal-title":"Current Protocols in Molecular Biology"},{"key":"e_1_2_10_2_35_1","first-page":"28.5:28.5.1","article-title":"Eradication of Mycoplasma Contaminations from Cell Cultures","volume":"106","author":"Uphoff C. C.","year":"2014","journal-title":"Current Protocols in Molecular Biology"},{"key":"e_1_2_10_2_36_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms9083"},{"key":"e_1_2_10_2_37_1","doi-asserted-by":"crossref","first-page":"II:2.5A:2.5A.1","DOI":"10.1002\/0471142727.mb0205as51","article-title":"Agarose Gel Electrophoresis","volume":"51","author":"Voytas D.","year":"2000","journal-title":"Current Protocols in Molecular Biology"},{"key":"e_1_2_10_2_38_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2015.12.035"},{"key":"e_1_2_10_2_39_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.3715"},{"key":"e_1_2_10_2_40_1","unstructured":"Retrieved fromhttps:\/\/www.nature.com\/nbt\/journal\/v34\/n12\/abs\/nbt.3715.html\u2010supplementary\u2010information."},{"key":"e_1_2_10_3_2_1","unstructured":"https:\/\/benchling.com."},{"key":"e_1_2_10_3_4_1","unstructured":"https:\/\/media.addgene.org\/data\/plasmids\/52\/52963\/52963\u2010attachment_IPB7ZL_hJcbm.pdf."},{"key":"e_1_2_10_3_6_1","unstructured":"https:\/\/portals.broadinstitute.org\/gpp\/public\/analysis\u2010tools\/sgrna\u2010design."},{"key":"e_1_2_10_3_8_1","unstructured":"https:\/\/www.idtdna.com\/PrimerQuest\/."},{"key":"e_1_2_10_3_10_1","unstructured":"https:\/\/tide\u2010calculator.nki.nl\/."}],"container-title":["Current Protocols in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/api.wiley.com\/onlinelibrary\/tdm\/v1\/articles\/10.1002%2Fcpmb.51","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpmb.51","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1002\/cpmb.51","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/pdf\/10.1002\/cpmb.51","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,8,31]],"date-time":"2023-08-31T15:18:46Z","timestamp":1693495126000},"score":1,"resource":{"primary":{"URL":"https:\/\/currentprotocols.onlinelibrary.wiley.com\/doi\/10.1002\/cpmb.51"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,1]]},"references-count":45,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2018,1]]}},"alternative-id":["10.1002\/cpmb.51"],"URL":"https:\/\/doi.org\/10.1002\/cpmb.51","archive":["Portico"],"relation":{},"ISSN":["1934-3639","1934-3647"],"issn-type":[{"value":"1934-3639","type":"print"},{"value":"1934-3647","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,1]]}}}