Fig 1.
Design of guide RNAs for CRISPR/Cas9 targeting of JCV.
A. Three gRNAs (TM1, TM2 and TM3) were designed at different positions within the coding region for JCV T-Ag (T) as shown. The T-Ag coding region begins at nucleotide (nt) 5013 of the 5130 nt circular Mad-1 JCV genome (NCBI Reference Sequence: NC_001699.1; Frisque et al, 1984) and proceeds counter clockwise to nt 2603. B. The sequence of the JCV genome at each of the three targeted sites (bold and red boxes) is given. Note that the sequence of the top strand is clockwise and antisense to the coding region of T-Ag since T-Ag is transcribed counter clockwise and shown on the bottom (sense strand). The position of the protospacer adjacent motif (PAM) sequences are shown in blue italics.
Fig 2.
Expression of gRNAs m1 and m2 knocks down T-Ag expression and effect T-Ag-induced activation of the JCV late promoter.
A. Cell extracts prepared in Fig 2A were analyzed by Western blot for expression of T-Ag, Cas9, and α-tubulin (loading control). B. The Western blots in Fig 2B were quantified using Bio-Rad Quantity One software and shown as a histogram normalized to T-Ag alone (lane 2). C. TC620 cells were transfected with JCVL-LUC reporter plasmid and expression plasmid for Cas9 with and without expression plasmids for T-Ag and each of the gRNAs shown in Fig 1 alone or in combination as indicated. Cells were harvested and luciferase activity was assayed as described in Materials and Methods. Activities were normalized to cells transfected with reporter plasmid alone (lane 1).
Fig 3.
A clonal derivative of SVG-A expressing Cas9 and gRNA m1 has reduced capacity to support JCV infection.
A. SVG-A cells were transfected with Cas9 or Cas9 plus gRNA m1 and stable clonal cell lines selected. Representative clones were selected and assayed for JCV infection (MOI = 1, 7 days post-infection): results from one clone with Cas9 alone and one clone with Cas9 plus gRNA m1 (c8) relative to parental SVG-A cells. Viral infection was assessed by Western blot for VP1 and agnoprotein with α-tubulin as a loading control. B. Viral loads in the culture supernatants from the experiment in Panel A were quantified using Q-PCR and are shown as copy number. C. SVG-ACas9 and SVG-ACas9m1c8 were assayed for Cas9 expression by Western blot with α-tubulin used as a loading control. D. TC620 cells were transfected with expression plasmid for FLAG-tagged Cas9 and immunocytochemistry performed with anti-FLAG antibody as described in Materials and Methods. Nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI).
Fig 4.
Direct demonstration of T-Ag gene cleavage after transient transfection of Cas9 and JCV-specific gRNA.
Mouse BsB8 and hamster HJC-2 cells, which both contain integrated JCV T-Ag gene, were transfected with expression plasmids for Cas9 and the gRNAs in various combinations as indicated and genomic DNA amplified using JCV-specific primers. A. Diagram of the T-Ag gene indicating the positions of the PCR primers and the expected cleavage points and the expected lengths of resulting fragments of the T-Ag gene. B. The T-Ag gene from transfected BsB8 cells was amplified by PCR, electrophoresed on an agrose gel and labeled with ethidium bromide. C. The T-Ag gene from transfected HJC-2 cells was amplified by PCR, electrophoresed on an agrose gel and labeled with ethidium bromide. The images in panels B and C are inverted for clarity of presentation.
Fig 5.
Stable derivatives of HJC-2 cells expressing doxycycline-inducible Cas9 show InDel mutations of the T-Ag gene upon transduction with lentiviruses expressing JCV-specific gRNAs and doxycycline induction.
A. HJC-2 cells expressing doxycycline-inducible Cas9 were transduced with lentiviruses expressing JCV-specific gRNAs and treated with and without doxycycline as described in Materials and Methods. Total genomic DNA was extracted and regions of the T-Ag were amplified by PCR, cloned into pCR4-TOPOTA vector and sequenced. B. The Surveyor assay was used to detect the presence of mutations in PCR products derived from HJC-2 cells expressing Cas9 and transduced by lentiviral vectors for each of the gRNAs (m1, m2 and m3). PCR products were denatured and hybridized by gradual cooling as described in Materials and Methods. Hybridized DNA was digested with SURVEYOR nuclease to cut heteroduplex DNA and samples were resolved on a 2% agarose gel together with equal amounts of control samples treated in parallel but derived from HJC-2 cells expressing Cas9 but not transduced by lentiviral vectors endocing gRNAs (m1 Con, m2 Con, m3 Con).
Fig 6.
Stable derivatives of HJC-2 cells expressing doxycycline-inducible Cas9 show ablation of T-Ag expression upon transduction with lentiviruses expressing JCV-specific gRNAs.
HJC-2 stable cell clones expressing doxycycline-inducible Cas9 were transduced with lentiviral vectors for each of the three gRNAs as described in Material and Methods. After 24 hours, the transduced cells were treated with and without 2 μg/ml doxycycline and after another 48 hours harvested and expression of T-Ag and Cas9 analyzed by Western blot with α-tubulin as a loading control.
Fig 7.
Stable derivatives of HJC-2 cells expressing doxycycline-inducible Cas9 show reduced colony formation upon transduction with lentiviruses expressing JCV-specific gRNAs.
A. HJC-2 stable cell clones expressing doxycycline-inducible Cas9 were transduced with lentiviruses expressing m1, m2 and m3 gRNAs in various combinations as indicated, plated, treated with or without doxycycline, and assessed for colony formation as described in Materials and Methods. Results are shown as histograms of the total numbers of colonies obtained with the error bars representing one standard deviation calculated from replicate colony counts. B. Photograph of a representative experiment showing two dishes from panel A after methylene blue staining.
Fig 8.
Stable derivatives of SVG-A cells expressing Cas9 and gRNAs show no InDel mutations in off-target genes.
The SURVEYOR assay was used to detect the presence of mutations in PCR products derived from SVG-A cells expressing Cas9 and gRNAs. m 1 (A), m2 (B) and m3 (C). Human cellular genes with the highest degree of homology to each motif were identified by BLAST search at the NCBI website (http://www.ncbi.nlm.nih.gov/). For each motif, PCR product was amplified from the top three genes with the highest degree of homology and examined for InDel mutations using the SURVEYOR assay as described in Materials and Methods. Amplification of T-Ag was the positive control in each panel. A. For motif m1, we amplified M12 (NM_017821, human RHBDL2 rhomboid, veinlet-like 2 (Drosophila), Gene ID: 54933, NCBI Ref Seq: NC_000001.11, >gi|568815597:c38941830-38885806), M17 (NM_001243540, human KIAA1731NL, Gene ID: 100653515, NCBI Ref Seq: NC_000017.11, >gi|568815581:78887721–78903217) and M19 (NM_016252, human BIRC6 baculoviral IAP repeat containing 6, Gene ID: 57448, NCBI Ref Seq: NC_000002.12); B. For motif m2, we amplified M21 (NM_012090, human MACF1 microtubule-actin crosslinking factor 1, Gene ID: 23499, NCBI Ref Seq: NC_000001.11, >gi|568815597:39084167–39487138), M23 (NM_005898, human CAPRIN1 cell cycle associated protein 1, Gene ID: 4076, NCBI Ref Seq: NC_000011.10, >gi|568815587:34051683–34102610), M24 (NM_024562, human TANGO6 transport and Golgi organization 6 homolog (Drosophila), Gene ID: 79613, NCBI Ref Seq: NC_000016.10, >gi|568815582:68843553–69085482); C. For motif m3, we amplified M31 (NM_001048194, human RCC1 regulator of chromosome condensation 1, Gene ID: 1104, NCBI Ref Seq: NC_000001.11, >gi|568815597:28505943–28539196), M32 (NM_004673, human ANGPTL1 angiopoietin-like 1, Gene ID: 9068, NCBI Ref Seq: NC_000001.11, >gi|568815597:c178871353-178849535), M33 (NM_174944, human TSSK4 testis-specific serine kinase 4, Gene ID: 283629, NCBI Ref Seq: NC_000014.9, >gi|568815584:24205530–24208248).