Current protocols of molecular biology: CRISPR/Cas9-Mediated Genome Editing in Epstein-Barr Virus-Transformed Lymphoblastoid B-Cell Lines.

Authors:
Jiang S, Wang LW, Walsh MJ, Trudeau SJ, Gerdt C, Zhao B, Gewurz BE.
In:
Source:
Publication Date: (2018)
Issue: 121: 31
Research Area:
Immunotherapy / Hematology
Cells used in publication:
LCL
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofectorâ„¢ X-Unit
Experiment

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Abstract

Epstein-Barr 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-expression 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-guide RNAs (sgRNAs), or dual-targeting sgRNAs, via lentiviral transduction of LCLs that stably express Cas9 protein. CRISPR/Cas9 editing allows LCL loss-of-function studies, including knock-out of protein-coding genes or deletion of DNA regulatory elements, and can be adapted for large-scale screening approaches. Low transfection efficiencies are a second barrier to performing CRISPR editing in LCLs, which are not typically lipid-transfectable. 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-cell lines, including Burkitt lymphoma and diffuse large B-cell lymphoma cells, and are highly reproducible.