CRISPR/Cas9-mediated Knockout and In Situ Inversion of ORF57 Gene from All Copies of The KSHV Genome in BCBL-1 Cells

Kaposi's sarcoma-associated herpesvirus (KSHV)-transformed primary effusion lymphoma cell lines contain ~70-150 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 of 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 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 ORF57 gene in iSLK/Bac16 and HEK293/Bac36 cells which contain only a few copies of KSHV genome. The ORF57-KO genome was stable in iSLK/Bac16 cells and upon lytic induction, could be partially rescued to express viral lytic gene ORF59 and production of infectious virions. Together, the technology developed in this study has paved the way to express two separate gRNAs and 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 could be applied to knock out the ORF57 gene from all ~100 copies of KSHV genome in PEL cells by co-expression of two gRNAs and Cas9 from a single expression vector in combination of single cell cloning. The gene knockout efficiency in this system could be evaluated rapidly using a direct cell PCR screening. The current CRISPR/Cas9 technology could also mediate ORF57 inversion in situ in the targeted site of KSHV genome. The successful rescue of viral lytic gene expression and infectious virion production from the ORF57-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 virus genome and to understand their contributions to viral genetics and virus life cycle. This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

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