CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ∼70 to 150 copies of episomal KSHV genomes per cell and have been widely used for studying the mechanisms of KSHV latency and lytic reactivation. Here, we report the first complete knockout (KO) of viral ORF57 gene from all ∼100 copies of KSHV genome per cell in BCBL-1 cells. This was achieved by a modified CRISPR/Cas9 technology to simultaneously express two guide RNAs (gRNAs) and Cas9 from a single expression vector in transfected cells in combination with multiple rounds of cell selection and single-cell cloning. CRISPR/Cas9-mediated genome engineering induces the targeted gene deletion and inversion in situ. We found the inverted ORF57 gene in the targeted site in the KSHV genome in one of two characterized single cell clones. Knockout of ORF57 from the KSHV genome led to viral genome instability, thereby reducing viral genome copies and expression of viral lytic genes in BCBL-1-derived single-cell clones. The modified CRISPR/Cas9 technology was very efficient in knocking out the ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells, where each cell contains only a few copies of the KSHV genome. The ORF57 KO genome was stable in iSLK/Bac16 cells, and, upon lytic induction, was partially rescued by ectopic ORF57 to express viral lytic gene ORF59 and produce infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and the Cas9 enzyme simultaneously in the same cell and could be efficiently applied to any genetic alterations from various genomes, including those in extreme high copy numbers. IMPORTANCE This study provides the first evidence that CRISPR/Cas9 technology can be applied to knock out the ORF57 gene from all ∼100 copies of the KSHV genome in primary effusion lymphoma (PEL) cells by coexpressing two guide RNAs (gRNAs) and Cas9 from a single expression vector in combination with single-cell cloning. The gene knockout efficiency in this system was evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology also mediated ORF57 inversion in situ in the targeted site of the KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57 knockout (KO) genome further reiterates the essential role of ORF57 in KSHV infection and multiplication. This modified technology should be useful for knocking out any viral genes from a genome to dissect functions of individual viral genes in the context of the virus genome and to understand their contributions to viral genetics and the virus life cycle.

Quantitative Proteomics Analysis of Lytic KSHV Infection in Human Endothelial Cells Reveals Targets of Viral Immune Modulation

“…CRISPR has recently been successfully used in other human herpesvirus infections: HSV-1 ( Suenaga et al., 2014 ), HCMV ( King and Munger, 2019 ), and EBV ( van Diemen et al., 2016 ). During preparation of this manuscript, the CRISPR-mediated modification of KSHV ORF57 from PEL cell lines was also reported ( BeltCappellino et al., 2019 ). The main challenge of using CRISPR in KSHV infection is that multiple viral genomes need to be targeted within the same cell, reducing the likelihood of generating a complete viral gene knockout.…”

Designer nucleases to treat malignant cancers driven by viral oncogenes

“…As the cell lines were not KSHV-dependent, effects on growth could not be observed, but may be a promising approach to deplete KSHV genomes. The loss of genomes was also observed when engineering CRISPR knockouts of the ORF57 gene in PEL cells [ 85 ], which further suggests that simply targeting the episome could result in nuclease-mediated degradation (Fig. 1 ).…”

Targeting the Kaposi’s sarcoma-associated herpesvirus genome with the CRISPR-Cas9 platform in latently infected cells

“…Accordingly, caution must be taken when performing functional studies that target the viral genome, since the reduction of viral DNA load could result from incomplete repair rather than indicating a specific functional role of the targeted sequence. In fact, successful knockout of the orf57 lytic gene from all copies of KSHV genome in PEL cells has been recently achieved, yet unexpectedly, it led to reduction of viral genome copies, likely due to incomplete repair [ 42 ]. Our study employed BAC16-mCherryORF45-infected cells, which enable continuous tracking of induction of lytic replication through microscopic detection of the immediate-early lytic protein mCherryORF45 [ 24 ].…”