Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus–host interactions. CRISPR systems enable focused or systematic, genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, CRISPR is becoming an essential tool in studies of the host factors important for viral pathogenesis. Here, we review the use of CRISPR–Cas9 for the loss-of-function analysis of host dependency factors. We focus on the use of CRISPR-pooled screens for the systematic identification of host dependency factors, particularly in Epstein–Barr virus-transformed B cells. We also discuss the use of CRISPR interference (CRISPRi) and gain-of-function CRISPR activation (CRISPRa) approaches to probe virus–host interactions. Finally, we comment on the future directions enabled by combinatorial CRISPR screens.
Highlights
Introduction to CRISPRClustered regularly interspaced short palindromic repeats (CRISPR)–Cas are the RNA and protein-based adaptive immune system that protect bacteria and archaea against invading viruses and foreign nucleic acids [1,2]
The CRISPR locus is transcribed into pre-CRISPR RNA, which is processed by Cas proteins and accessory factors into mature crRNA [4]
These data are consistent with a model where Epstein–Barr virus (EBV)-induced BATF and Interferon Regulatory Factor 4 (IRF4) bind to a composite DNA site to recruit EBNA3A/C and polycomb repressor complexes that include the lymphoblastoid cell lines (LCL) dependency factor CTBP1
Summary
Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas are the RNA and protein-based adaptive immune system that protect bacteria and archaea against invading viruses and foreign nucleic acids [1,2]. CRISPR spacers are archived sequences, typically 23–44 base pairs in length, copied from phage or plasmid DNA that previously invaded the bacterium [3]. These sequences are interspersed between repeat sequences and are located adjacent to the CRISPR-associated (Cas) genes, which encode. TracrRNA and crRNA are typically fused into a single-guide RNA (sgRNA) transcript for eukaryotic genome engineering. HNH nuclease domains create a double-strand break in the break crRNA-paired sequence, so long as so longisasathere is a protospacer adjacent (PAM). Custom sgRNAs contain 20 base pairs of crRNA sequence complementary to the target DNA site [9,17]. CRISPR editing could even be achieved with vaccinia virus, which replicates its DNA in the cytosol [29]
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