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. 2019 Oct 15;93(21):e00628-19.
doi: 10.1128/JVI.00628-19. Print 2019 Nov 1.

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

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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

Andrew BeltCappellino et al. J Virol. .
Free PMC article

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.

Keywords: BCBL-1; CRISPR; Cas9; Kaposi's sarcoma-associated herpesvirus; ORF57; PEL cells; gene inversion; knockout.

Figures

FIG 1
FIG 1
Generation of KSHV ORF57 knockout (KO) in Bac36 cells using a common CRISPR/Cas9 technology. (A) Diagram of wild-type (WT) KSHV ORF56-ORF57 locus including the promoters (arrows with P) and a common polyadenylation site (pA) used for both ORF56 and ORF57 expression. Red boxes represent the Cas9/gRNA-targeted sites in both 5′ and 3′ regions of the ORF57 gene (nt 82069 to 83544) to induce ORF57 deletion. The resulted ORF57 KO genome retains the intact ORF56 ORF and the pA site for ORF56 expression. (B) The targeted sequences of gRNAs used for ORF57 KO within the KSHV genome along with the PAM sequences shown in red and their nucleotide positions in the reference KSHV genome (GenBank accession number U75698). (C and D) The CRISPR/Cas9-mediated KO efficiency of ORF57 gene from the KSHV genome in Bac36 cells transfected with the indicated plasmids expressing various combinations of single ORF57 gRNA. The efficiency of ORF57 KO was determined by PCR (C) on total cell DNA using Pr1 plus Pr2 primers flanking the deleted region of ORF57 gene (A) or by anti-ORF57 antibody Western blotting on total cell lysates 24 h after sodium butyrate (Bu) induction of ORF57 protein expression (D). Cellular β-tubulin served as a loading control.
FIG 2
FIG 2
Construction of the second-generation CRISPR/Cas9 vectors for expression of dual gRNAs. (A) Diagram showing construction of a CRISPR/Cas9 vector capable of simultaneous expression of two different gRNAs. First, the locus consisting of upstream U6 promoter (black arrow) and gRNA (red or green arrows) were amplified by PCR using flanking primers (black thin arrows). The resulting PCR product was inserted into a vector containing gRNA 1 or gRNA 2 using XbaI and KpnI sites introduced by PCR amplification. The final vector structure contains the two separate gRNAs each under the control of a U6 promoter followed by Cas9-Puro locus under the control of CBh (chicken β-actin hybrid) promoter. (B) Validation of functionality and potency of the second-generation gRNA vectors to facilitate ORF57 KO in Bac36 cells as determined by PCR using a Pr1 plus Pr2 primer set described in Fig. 1A. WT, wild type; KO, knockout.
FIG 3
FIG 3
KSHV ORF57 KO in BCBL-1 cells. (A) Work flow showing multiple rounds of BCBL-1 cell transfection with the second generation of gRNA vectors followed by 3 days of puromycin selection. After achieving a detectable level of ORF57 KO, the cells were subjected to prolong puromycin selection and single-cell cloning by limited dilution in a 96-well plate. The obtained clones were expanded and used for PCR screen. (B) The KSHV ORF56-ORF57 locus, targeted sites by gRNAs (red squares), and PCR screening primers (black arrows below the diagram). (C) The efficiency of ORF57 KO in BCBL-1 cells after 1, 2, and 4 selection cycles as determined by PCR using Pr1 plus Pr2 primers. (D and E) ORF57 KO and PCR screening of isolated BCBL-1 single-cell clones derived from gRNA 2 plus 4 vector stable transfection by using Pr1 plus Pr2 (D) or Pr1 plus Pr3 (E) primers as described in panel B. WT, wild type ORF57 gene product; KO, knockout ORF57 gene product; B-1, parental BCBL-1 cell population.
FIG 4
FIG 4
Selection of BCBL-1 ORF57 KO single-cell clones expressing ORF50 (RTA, a viral transactivator), Cas9, and ORF56. (A) Western blot analysis of ORF57, ORF50, and Cas9 protein expression in selected BCBL-1 ORF57 KO clones after viral reactivation with sodium valproate (VA) for 24 h. Cellular β-tubulin served as a protein loading control. (B) Expression of ORF57 protein (red) as detected by immunofluorescence staining using anti-ORF57 antibody in BCBL-1 WT and ORF57 KO cells (clone 39) after KSHV reactivation with VA for 24 h. The cell nuclei were counterstained by DAPI. Bar, 10 μm. (C) RT-PCR analysis of ORF56 expression in ORF57 KO single-cell clone 39. Total RNA was prepared after viral reactivation with sodium valproate (VA) for 24 h, reverse transcribed in the presence (+) or absence (−) of reverse transcriptase (RT), and PCR amplified with ORF56-specific primer pair (see Table S1 in the supplemental material for details). Total RNA extracted from parental BCBL-1 cells (WT) or from a BCBL-1 cell stably transfected with a Cas9-only vector served as a control for ORF56 detection. GAPDH served as an internal RNA loading control.
FIG 5
FIG 5
Verification of the KSHV genome with ORF57 deletion in BCBL-1 ORF57 KO clones by Southern blotting. (A) Diagram depicting ORF57 locus targeted by gRNAs 2 and 4 and HindIII restriction sites. Horizontal black arrows represent the primers used in PCR screening. Below are DNA fragments predicted from the WT or ORF57 KO genome after HindIII digestion. A blue arrow represents an oligonucleotide probe used for Southern blotting. (B) Southern blot analysis of KSHV genomic DNA from original BCBL-1 cells (WT) or two separate ORF57 KO single-cell clones (#6 and #39) after HindIII digestion using a 32P-labeled oligonucleotide probe oVM9 (Pr1) shown in panel A. (C) Sequencing of the guide RNA-mediated deletion junction in the ORF57 KO genome. PCR products were obtained with the primer pair of Pr1 plus Pr2 from the single-cell clone 39 DNA and then TA cloned and sequenced. Chromatographs of two representative TA-clones show the sequence junction at the target site (TS) of guide RNA-mediated ORF57 deletion without or with additional “A” insertion. The numbers above the DNA junctions mark the nucleotide positions in the KSHV genome (GenBank accession number U75698).
FIG 6
FIG 6
Identification of guide RNA-mediated ORF57 gene inversion (INV) in the KSHV genome in ORF57 KO clone 6 cells. Diagrams of the KSHV WT genome (A) and an inverted ORF57 gene in the clone 6 virus genome (B). Predicted DNA fragments after HindIII digestion are shown below for both virus genomes. Horizontal black arrows below the diagrams are two backward oligonucleotide primers Pr3 and Pr2 for the WT genome (A), but the primer Pr3 can be used in PCR as a forward primer with the primer Pr2 for an inverted ORF57 gene in the clone 6 virus genome (B). Oligonucleotide oVM9 described in the legend for Fig. 5 and oVM65 in blue were diagramed at relative positions in the viral genome. Oligo oVM65 was used for Southern blot analysis in this figure. (C) Southern blot analysis of the inverted ORF57 gene mediated by CRISPR/Cas9 technology. The same membrane used in the Fig. 5B Southern blot was reprobed with an ORF57 internal probe, oVM65. See the predicted size of HindIII fragments detected by this probe in panels A and B. (D) Detection and sequencing of the inverted ORF57 gene in the clone 6 cells by PCR using primers Pr3 plus Pr2 shown in panel B. The PCR products from the KSHV ORF57 single clone 6 cells were TA cloned and sequenced. (E) The primer Pr3-derived sequence chromatograph confirmed the junction sequences between the inverted target site (TS) of gRNA 2 and the TS of gRNA 4, together with the inverted ORF57 coding region (purple).
FIG 7
FIG 7
ORF57 KO induces KSHV genome instability and reduces viral gene expression in BCBL-1 cells. (A) Estimated copy numbers of the KSHV WT and ORF57 KO genomes per BCBL-1 cell. Total DNA isolated from BCBL-1 cells carrying a WT or ORF57 KO (clone 6) KSHV genome was analyzed by whole-genome sequencing (WGS). The copy number of the KSHV genome in each cell clone was then calculated as described in Materials and Methods and illustrated as the mean reads depth profile of each ORF crossing the entire genome. (B) Relative KSHV genome DNA level determined by qPCR. Total DNA from WT KSHV genome and ORF57 KO genome (clone 6) cells was quantified for ORF59 DNA by qPCR. Host lncFXBO9-11 DNA served as an internal control for normalization. (C) Viral read coverage profiles from the WT genome to the ORF57 KO genome illustrated by IGV. KSHV BCBL-1 WT genome sequence (GenBank accession number MN205539) from the parental BCBL-1 cells was compared with the ORF57 KO genome sequence isolated from the clone 6 cells. Shown below are the zoomed alignment tracks with the reads colored according to pair orientations for the inverted sites identified in KSHV BCBL-1 genome when compared with the reference KSHV genome (GenBank accession number U75698). The inverted ORF57 locus in the ORF57 KO is shown on the right. (D and E) Effect of ORF57 KO and inversion on viral genome expression in ORF57 KO cells. Parental BCBL-1 WT and two stable ORF57 KO single-cell clones were reactivated for viral lytic infection by 1 mM sodium valproate for 24 h before total cell lysates and RNA were extracted separately. The expression of individual viral genes from each cell sample was examined either by Western blotting (D) for selected viral proteins or by Northern blotting (E) for indicated viral gene transcripts.
FIG 8
FIG 8
Detrimental effect of CRISPR/Cas9-mediated ORF57 KO on KSHV genome expression and virion production in iSLK/Bac16 cells containing a stably transfected KSHV-GFP genome. (A to C) PCR screen of ORF57 KO efficiency using flanking primers Pr1 plus Pr2 (Fig. 1A) in the untransfected iSLK/Bac16 cells (none) or the cells transfected by a Cas9-only empty vector (vector) or a gRNA 2 plus 4 expression vector (gRNA2 + 4) (A). Characterization of the ORF57 KO genome for expression of ORF50, ORF57, and ORF59 in the selected single-cell clones. The iSLK/Bac16 cells with or without transfection by a Cas9-only empty vector (vector) or a gRNA 2 plus 4 expression vector (gRNA2 + 4) as described in the legend for Fig. 3 were subjected to single-cell cloning and screening before induction of KSHV lytic replication by doxycycline and sodium butyrate. Total cell lysates and RNA before and after selection of single cell clones and viral lytic induction were prepared for Western blot analyses of KSHV ORF50 and ORF57 (B) and for RT-qPCR analysis of ORF59 (C). Cells without transfection (none) or transfected with a Cas9-only vector (vector) before selection served as controls and cellular β-tubulin served as a loading control (B). (D) WT and ORF57 KO cells with lytic induction by Dox plus Bu for 24 h were examined for GFP expression level in association with KSHV genome copies. The expression of ORF57 was examined by anti-ORF57 IF staining to determine ORF57 KO efficiency. Bar, 100 μm. Effect of ORF57 KO on infectious KSHV production. (E and F) Titration of cell-free virus production from Dox- plus Bu-induced iSLK/Bac16 cells carrying a KSHV WT or ORF57 KO (clone 2G1) genome was carried out in HEK293T cells by using the corresponding cell culture medium collected 5 days after viral lytic induction. The newly infected HEK293T cells expressing GFP as indications of GFP-KSHV virus infection were imaged by direct fluorescence (E) and then counted by FACS analysis (F) 48 h after infection. The percentages of GFP+ cells over total number of cells were calculated based on the cell counts in the Q4 region (F).
FIG 9
FIG 9
Rescue of infectious KSHV production from iSLK/Bac16 cells containing an ORF57 KO KSHV genome. (A and B) Rescue of ORF59 expression from an ORF57 KO genome in the clone 2G1 cells by ectopic expression of ORF57. The 2G1 single-cell clone derived from iSLK/Bac16 cells containing an ORF57 KO KSHV-GFP genome was used for transfection of an empty (FLAG only) or ORF57-FLAG expression vector. After transfection, the virus was reactivated with Dox plus Bu treatment for 24 h. The expression of ORF59 or ectopic ORF57-FLAG was monitored by IF staining by using an anti-ORF59 specific antibody alone (A) or in combination with an anti-FLAG antibody for ORF57 expression (B). The WT and ORF57 KO 2G1 cells without transfection served as controls (A). Bar, 100 μm in panel A and 20 μm in panel B. (C and D) Ectopic expression of ORF57-FLAG in ORF57 KO 2G1 cells rescues the production of infectious virions. The 2G1 cells carrying an ORF57 KO genome were transfected with an empty (FLAG only) or ORF57-FLAG-expressing vector. The virus replication was induced by Dox plus Bu treatment, and the culture supernatant containing cell-free virions was collected 5 days after induction. The production of infectious virions was determined by inoculating HEK293T cells with the collected supernatant. The number of newly infected GFP+ cells was determined 48 h after infection using live fluorescence imaging (C) or by FACS analysis (D). The 2G1 cells with transfection of an empty FLAG vector served as negative controls. Bar, 100 μm.

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