<i>CCR5</i> Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

<div><p>CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced <i>CCR5</i> via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4⁺ cells yields high frequencies of <i>CCR5</i> gene disruption. <i>CCR5</i> gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over <i>CCR5</i> gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of <i>CCR5</i> via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1.</p></div

CCR5-/CR2 cells confer resistant to R5-tropic viruses and exhibits selective advantage during R5-tropic HIV-1 infection.

<p>A. HIV-1 replication in sorted CCR5-/CR2 and CCR5+/GF1 cells infected with X4-tropic HIV-1 Bru3. B. HIV-1 replication in sorted CCR5-/CR2 and CCR5+/GF1 cells infected with R5-tropic HIV-1 Bru-Yu2. C. FACS analysis of cell surface expression of CD4 and CCR5 in mixtures of CCR5-/CR2 and parental CEMss-CCR5 cells at 6 and 18 days post-infection with X4-tropic HIV-1 Bru3 (middle panel), R5-tropic HIV-1 Bru-Yu2 (right panel), or no virus control (left panel). D. <i>CCR5</i> gene analysis on uncleaved versus cleaved bands in parental CEMss-CCR5 cells (line 1) or CCR5-/CR2 and parental CEMss-CCR5 mixture at 0, 6 and 18 days post infection with X4 tropic HIV-1 Bru3 (lines 2 to 4) or R5-tropic HIV-1 Bru-Yu2 (lines 5 to 7) by T7EI assay. DPI: days post infection.</p

<i>CCR5</i> gene disruption by RNA-guided Cas9 endonuclease in transduced TZM.bl cells.

<p>A. Schematic diagrams of lentiviral transfer vectors containing Cas9 endonuclease or sgRNAs CR1, CR2, Cr3 and GF1. HA: HA epitope tag; NLS: nuclear localization signal; PGK: promoter sequence derived from phosphoglycerate kinase-1; U6: promoter sequence derived from polymerase III U6. B. Cell surface expression of CCR5 on TZM.bl cells co-transduced with lentiviral vectors expressing Cas9-HA-NLS and sgRNAs CR1, CR2, CR3 or GF1. Transduced (open curves) and mock-transduced (shaded curves) cells were stained with anti-CCR5 antibody followed by FACS analysis. C. FSC and SSC analysis mock-transduced TZM.bl cells and TZM.bl cells transduced with lentiviral vectors expressing Cas9-HA-NLS fusion protein and sgRNAs CR1, CR2, CR3 or GF1, respectively. Percentages of gated (live) cells are shown at the lower and left corner of the figures. D. <i>CCR5</i> gene disruption analysis by T7EI assay using prime pairs CR1/F990-CR1/R1750 and CR2/3F593-CR2/3R1254 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115987#pone.0115987.s003" target="_blank">S1 Table</a>). E. <i>CCR5</i> gene disruption analysis by T7EI assay using prime pair CR1/2/3F2559-CR1/2/3R3893 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115987#pone.0115987.s003" target="_blank">S1 Table</a>).</p

<i>CCR5</i> gene disruption by RNA-guided Cas9 endonuclease in transduced CEM ss-CCR5 cells.

<p>A. Cell surface expression of CCR5 on CEMss-CCR5 cells co-transduced with lentiviral vectors expressing Cas9-HA-NLS and one of sgRNA (CR2 or GF1) as compared to mock-transduced CEMss-CCR5 cells. B. FSC and SSC analysis mock-transduced CEMss-CCR5 cells and CEMss-CCR5 cells transduced with lentiviral vectors expressing Cas9-HA-NLS fusion protein and sgRNAs CR2 or GF1, respectively. Percentages of gated (live) cells are shown at the lower and right corner of the figures. C. <i>CCR5</i> gene disruption analysis in mock-transduced CEMss-CCR5 cells or CEMss-CCR5 cells co-transduced with lentiviral vectors expressing Cas9-HA-NLS and one of sgRNAs (CR2 or GF1) by T7EI assay. D. Mean and median values of fluorescence intensity of cell surface expression of CD4, CXCR4 and CCR5 in sorted CCR5-/CR2 and CCR5+/GF1 cells as compared to parental CEMss-CCR5 cells. E. Relative fluorescent intensity of DDAO-labeled CCR5-/CR2 and CCR5+/GF1 cells as compared to parental CEMss-CCR5 cells at 0, 12, 24 and 36 hours post culture.</p

<i>CCR5</i> gene disruption by RNA-guided Cas9 endonuclease in sorted CCR5-/CR1, CCR5-/CR2 and CCR5-/CR3 cells by Sanger sequencing analysis.

<p>A-C. Cell surface expression of CCR5 on sorted CCR5-/CR1 (A), CCR5-/CR2 (B) and CCR5-/CR3 (C) TZM.bl cells as compared to mock-transduced TZM.bl cells. D-F. Representative Sanger sequencing of CCR5 target sites by CR1 (D), CR2 (E) or CR3 (F) in co-transduced TZM.bl cells. The full list of Sanger sequencing of CCR5 target sites by CR1, CR2 or CR3 is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115987#pone.0115987.s002" target="_blank">S2 Fig</a>. For each <i>CCR5</i> site targeted by CR1 (D), CR2 (E) or CR3 (F), the upper panel shows representative Sanger sequencing of targeted CCR5 sequences amplified by a prime pair that covers both the endogeous <i>CCR5</i> gene and transgene. The lower panel shows representative Sanger sequencing of targeted <i>CCR5</i> sequences by a prime pair that only covers the endogenous <i>CCR5</i> gene.</p

<i>CCR5</i> gene disruption in CEMss-CCR5 cells transduced with a single lentiviral vector expressing both CR2 sgRNA and Cas9.

<p>A. Schematic diagrams of a lentiviral transfer vector pRRL-CR2 sgRNA-Cas9/EGFP. Cas9: Cas9 endonuclease; CR2 sgRNA: single guided RNA CR2; HA: HA epitope tag; NLS: nuclear localization signal; U6: polymerase III U6 promoter sequence; EF1α: promoter sequence derived from elongation factor 1α; 2A: 2A self cleaving peptide sequence; and EGFP: sequence encoding enhanced green fluorescent protein. B. FSC and SSC analysis in mock-transduced CEMss-CCR5 cells and CEMss-CCR5 cells transduced with a single lentiviral vector pRRL-CR2 sgRNA-Cas9/EGFP. Percentages of gated (live) cells are shown at the lower and right corner of the top-right and top-left panels. CCR5 and EGFP analysis in mock-transduced CEMss-CCR5 cells and CEMss-CCR5 cells transduced with a single lentiviral vector pRRL-CR2 sgRNA-Cas9/EGFP. C. <i>CCR5</i> gene disruption analysis in mock-transduced CEMss-CCR5 cells or CEMss-CCR5 cells transduced with a single lentiviral vector pRRL-CR2 sgRNA-Cas9/EGFP by T7EI assay.</p

T7 endonuclease I (T7EI) analysis of potential off-target loci.

<p>A. T7EI analysis of three potential off-target loci (AKAP9, ULK1 and MED16) to CR2 as well as on-target locus CCR5. “–”: genomic DNA isolated from mock-transduced TZM.bl; “+”: genomic DNA isolated from sorted CCR5-/CR2 cells. D21 and D84: genomic DNA samples isolated from sorted CCR5-/CR2 cells at 21 and 84 days post transduction. B. T7EI analysis of nine potential off-target loci (SH2D5, ASB9P1, PRRT1, LINC00265, ENDOV, NR2F1, ASB9, CLPP and SOBP) to CR3 as well as on-target locus CCR5 control. “–”: genomic DNA isolated from parental TZM.bl; “+”: genomic DNA isolated from sorted CCR5-/CR3 cells. D21 and D84: genomic DNA samples isolated from sorted CCR5-/CR3 cells at 21 and 84 days post transduction.</p

Screening for XMRV in patient PBMCs by PCR.

<p>PCR products were analyzed on agarose gels containing ethidium bromide. (A) Representative gels for non-nested <i>env</i> (top panel) and non-nested <i>gag</i> (bottom panel) PCRs are shown containing a set of three replicates for each of 5 HIV-1⁺ patient samples. A yellow arrow indicates the sole PCR band, from patient 103219, found to be comprised of XMRV DNA by sequencing. (B) A representative gel for nested <i>env</i> PCR is shown for the same 5 HIV-1⁺ patient samples depicted in (A). Vertical black arrows in (A) and (B) indicate lanes from patient 103219 containing either (A, bottom panel) a band comprised of XMRV sequence or (B) a band of the expected mobility for the target sequence. (m) 100 base pair molecular weight marker, (1∶10⁴) DNA from one infected cell diluted in DNA from 10⁴ uninfected cells used as template for positive control.</p

Summary of XMRV screening results.

a<p>Fractions are: number of subjects scoring positive/total number of subjects screened.</p>b<p>Ab, antibody.</p

Detecting murine DNA by IAP PCR.

<p>PCR products were analyzed on 1.5% agarose gels containing ethidium bromide. (A) Sensitivity of the IAP PCR assay was determined by performing PCRs on titrations of EL4 murine cell line DNA in a background of 200 ng LNCaP DNA. One murine cell equivalent (1 eq) indicates 6 pg of EL4 DNA. XMRV-infected LNCaP (iLNCaP) and uninfected LNCaP (uLNCaP) were included as controls. (B) Screening results for 17 HIV-1⁺ patient samples. Arrow points to sample 103219, which tested positive for XMRV by non-nested gag PCR. (m) 100 base pair molecular weight marker, (EL4) 6 pg of murine EL4 cell line DNA without a background of human DNA.</p