Modulating Gene Expression in Epstein‐Barr Virus (EBV)‐Positive B Cell Lines with CRISPRa and CRISPRi

Abstract

Epstein-Barr virus (EBV) transforms small resting primary B cells into large lymphoblastoid cells which are able to grow and survive in vitro indefinitely. These cells represent a model for oncogenesis. In this unit, variants of conventional clustered regularly interspaced short palindromic repeats (CRISPR), namely the CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) methods, are discussed in the context of gene regulation at genomic DNA promoter and enhancer elements. Lymphoblastoid B cell lines (LCLs) stably expressing nuclease-deficient Cas9 (dCas9)-VP64 (Cas9 associated with CRISPRa) or dCas9-KRAB (Cas9 associated with CRISPRi) are transduced with lentivirus that encodes a single guide RNA (sgRNA) that targets a specific gene locus. The ribonucleoprotein complex formed by the dCas9 molecule and its cognate sgRNA enables sequence-specific binding at a promoter or enhancer of interest to affect the expression of genes regulated by the targeted promoter or enhancer. © 2018 by John Wiley & Sons, Inc.

Epstein-Barr Virus Induced Cytidine Metabolism Roles in Transformed B-Cell Growth and Survival

“…To study the functional significance of the EBNA and NF-κB-bound CTPS1 intragenic region in CTPS1 transcription regulation, we used CRISPR-interference (CRISPR-i) ( 35 ). GM12878 LCLs with endonuclease dead Cas9 (dCas9) fused to a KRAB transcription repressor domain were transduced with lentiviruses that express control sgRNA versus one of the 12 sgRNAs indicated in Fig.…”

“…52963; Addgene). Lentiviruses were prepared by transfecting HEK293T cells and used to transduce LCLs stably expressing a dCas9-KRAB fusion protein for CRISPR-i ( 35 ).…”

Epstein-Barr Virus Induced Cytidine Metabolism Roles in Transformed B-cell Growth and Survival

“…To study the functional significance of the EBNA and NF-□B-bound CTPS1 intragenic region in CTPS1 transcription regulation, we used CRISPR-interference (CRISPR-i) (36). GM12878 LCLs with endonuclease dead Cas9 fused to a KRAB transcription repressor domain were transduced with lentiviruses that express control sgRNA versus one of the twelve sgRNAs indicated in Figure 4a .…”

“…To study the functional significance of the EBNA and NF--bound CTPS1 intragenic region in CTPS1 transcription regulation, we used CRISPR-interference (CRISPR-i) (36). Figure 4c).…”

CRISPR–Cas9 Genetic Analysis of Virus–Host Interactions

“…The biological significance of the vast majority of these EBNA-bound enhancers on target gene expression and B cell growth or survival remains to be tested. We recently used CRISPRi to interrogate the effect of EBNA-bound LCL enhancer deactivation in a proof-of-principle study [ 140 ]. Furthermore, multiple EBNA-bound enhancers often loop to a target gene promoter, and these can in theory be simultaneously targeted by multiplex sgRNA approaches, in which the expression of multiple sgRNAs enables the simultaneous deactivation of multiple enhancers [ 141 ].…”