Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: CoV-2 nsp1 promotes translational suppression and mRNA decay in vitro and in cells (A) HBB-nLuc reporter RNA was incubated with HEK293T translation extracts alone or in the presence of increasing concentrations of purified WT, R124A/K125A, or K164A/H165A nsp1. Translation of the reporter was then evaluated by luciferase assay and normalized to a glutathione S-transferase (GST) protein control. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001; one-sample t test versus hypothetical value of 1. The bars represent the mean value of the replicates and error bars represent standard deviation. (B) A primer extension assay was used to measure cleavage of the HBB-nLuc RNA in the presence of purified WT or mutant nsp1. Lane 1 (no IVT) shows nsp1 and HBB-nLuc incubation in primer extension buffer only, whereas lanes 2–4 show reactions incubated in the presence of translation extracts. Hash marks denote cleavage intermediates. (C and D) HEK293T cells were transfected with a GFP reporter plasmid alone or together with the indicated nsp1-expressing plasmids and then harvested for protein or RNA. GFP and nsp1 protein levels were measured by ⍺-GFP and ⍺-FLAG western blots, respectively, with vinculin used as a protein loading control (C). GFP mRNA was quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 then set to 1 (D). Each dot represents an independent experiment. ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. For (D), the bars represent the mean value of the replicates and error bars represent standard deviation. (E) HEK293T cells transfected with a GFP reporter plasmid alone or together with the indicated nsp1-expressed plasmids were subsequently treated with 5-μg/mL actinomycin D (ActD) and harvested at the time points indicated after ActD treatment. GFP mRNA was quantified by qRT-PCR and normalized to 18S rRNA, and the changes in GFP mRNA abundance are relative to the time point immediately before ActD treatment. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001; two-way ANOVA with Geisser-Greenhouse correction followed by Tukey’s multiple comparisons test versus “0”-h time point. See also and . The points represent the mean values of the replicates and error bars represent standard deviation.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 118-180 nsp1 , This paper , 175424 (Addgene).

Techniques: In Vitro, Incubation, Purification, Luciferase, Standard Deviation, Primer Extension Assay, Mutagenesis, Transfection, Plasmid Preparation, Expressing, Western Blot, Quantitative RT-PCR

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: The N-terminal and central domains of nsp1 are required for translational suppression and mRNA depletion (A) Schematic of the N-terminal 3xFLAG-Halo-tagged versions of WT and mutant nsp1. Amino acids 122–130 encompass the RNA destabilization domain, which was either deleted (Δ122–130) or replaced with a size-matched glycine linker (G-linker). Mutant Δ118–180 lacks the central and C-terminal domains, whereas Δ1–117 lacks the N-terminal domain. (B and C) HEK293T cells were transfected with a GFP reporter plasmid alone or together with plasmids containing WT or the indicated mutant nsp1 and then harvested for protein or RNA. GFP, and nsp1 protein levels were measured by ⍺-GFP and ⍺-FLAG western blots, respectively, with vinculin used as a protein loading control (B). GFP mRNA was quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 then set to 1 (C). Each dot represents an independent experiment. ∗∗∗∗ p ≤ 0.0001; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. See also Figure S2 . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 118-180 nsp1 , This paper , 175424 (Addgene).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Residue R99 located in the N-terminal domain plays key roles in nsp1-induced host shutoff (A and B) HEK293T cells were transfected with a GFP reporter plasmid alone or together with plasmids containing WT or the indicated mutant nsp1 and then harvested for protein or RNA. GFP and nsp1 protein levels were measured by ⍺-GFP and ⍺-FLAG western blots, respectively, with vinculin used as a protein loading control (A). GFP mRNA was quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 then set to 1 (B). Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗∗∗ p ≤ 0.0001; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. For (B), the bars represent the mean value of the replicates and error bars represent standard deviation. (C) HBB-nLuc reporter RNA was incubated with HEK293T translation extracts in the presence of 80 nM of purified WT or the indicated mutant nsp1 protein. Translation of the reporter was then evaluated by luciferase assay and normalized to levels from lysates incubated with 80 nM of a control GST protein. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (D) Primer extension assay to measure degradation of the HBB-nLuc RNA in the presence and absence of purified WT or mutant nsp1. Lanes 1 and 2 are controls lacking translation extract (no IVT) or nsp1, respectively. See also Figure S3 .

Article Snippet: pCDNA4TO-3XFLAG Halo delta 118-180 nsp1 , This paper , 175424 (Addgene).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Quantitative RT-PCR, Standard Deviation, Incubation, Purification, Luciferase, Primer Extension Assay

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Nsp1 N-terminal and central domain mutants are defective for ribosome and mRNA binding (A) HEK293T cells were transfected with plasmids expressing WT or the indicated mutant 3xFLAG-Halo-tagged nsp1. Nsp1 was immunoprecipitated (IP) using ⍺-FLAG beads and coIP of ribosomal proteins RACK1, RPS2, RPS3, and RPS24 was monitored by western blotting, with vinculin serving as a loading control. Input lanes contain 1/10 of the amount of protein used for the IPs. (B) Equilibrium binding measurements of fluorescently labeled WT (blue), R124A,K125A (red), and R99A (green) nsp1 to purified ribosomes. Data represent a total of 3 biological replicates. (C) HEK293T cells were co-transfected with HBB-nLuc and either a control plasmid or the indicated 3xFLAG-Halo-tagged nsp1 constructs. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating RNAs were extracted and nLuc mRNA was quantified by qRT-PCR. The mRNA values were then normalized to the values obtained from the empty vector control. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (D) HEK293T cells were co-transfected with a 3xFLAG-Halo-tagged nsp1 plasmid or empty vector control, together with a plasmid expressing either GFP with a 5′ stem loop (GFP+SL) or a control GFP lacking the stem loop (GFP). Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating GFP+SL or GFP mRNAs were quantified by qRT-PCR. The mRNA values were then normalized to those obtained from the empty vector control. The bars represent the mean value of the replicates and error bars represent standard deviation. (E) The levels of GFP+SL and GFP mRNA present in the input samples from (D) were quantified by qRT-PCR and normalized to 18S rRNA, with the level of GFP mRNA in cells lacking nsp1 (empty vector control) set to 1. Each dot represents an independent experiment. ∗∗ p ≤ 0.01; unpaired t test. See also , , and . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 118-180 nsp1 , This paper , 175424 (Addgene).

Techniques: Binding Assay, Transfection, Expressing, Mutagenesis, Immunoprecipitation, Western Blot, Labeling, Purification, Plasmid Preparation, Construct, Quantitative RT-PCR, Standard Deviation

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Protection from translational repression conferred by the CoV-2-leader sequence is selectively eliminated by nsp1 N-terminal and central domain mutants (A) HEK293T cells were co-transfected with a plasmid expressing CoV-2 leader-nLuc and either a control plasmid or the indicated 3xFLAG-Halo-tagged nsp1 construct. Nsp1 was immunoprecipitated using ⍺-FLAG beads, whereupon the co-immunoprecipitating RNAs were quantified by qRT-PCR. The mRNA values were then normalized to the mRNA values obtained from the empty vector control. Each dot represents an independent experiment. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. The bars represent the mean value of the replicates and error bars represent standard deviation. (B) HEK293T cells were transfected with either HBB-nLuc or CoV2L-nLuc together with control empty vector or the indicated nsp1 construct. Translation of HBB-nLuc or CoV2L-nLuc was measured by luciferase assay, and the fold change in luciferase activity was calculated relative to the empty vector control. Technical triplicate measurements were taken for each biological replicate. A total of at least three biological replicates were taken for each measurement. ∗ p ≤ 0.05, ∗∗ p ≤ 0.01; one-sample t test versus hypothetical value of 1. The bars represent the mean value of the replicates and error bars represent standard deviation. (C) CoV2L-nLuc mRNA was quantified from the above experiment by qRT-PCR and normalized to 18S rRNA, with the level of CoV2L-nLuc mRNA in cells lacking nsp1 then set to 1. Each dot represents an independent experiment. ∗∗ p ≤ 0.01; one-way ANOVA followed by Dunnett’s multiple comparisons test versus WT nsp1. See also Figure S6 . The bars represent the mean value of the replicates and error bars represent standard deviation.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 118-180 nsp1 , This paper , 175424 (Addgene).

Techniques: Sequencing, Transfection, Plasmid Preparation, Expressing, Construct, Immunoprecipitation, Quantitative RT-PCR, Standard Deviation, Luciferase, Activity Assay

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet: Model for how the N-terminal and central domains of nsp1 are critical for 40S ribosome association and preservation of leader-containing transcripts (A) All three nsp1 domains contribute to its interaction with the 40S ribosome. While the C-terminal domain interjects into the mRNA entry channel of the ribosome to block mRNA access, the N-terminal and central domains stabilize the interaction. When cellular mRNA encounters an nsp1-bound ribosome, it is translationally blocked and undergoes degradation. However, mRNA containing the CoV-2 leader sequence engages the N-terminal and central domains of 40S-bound nsp1 in a manner involving nsp1 residues R124, K125, and R99, leading to relief from translational repression. (B) Nsp1 mutants R124A/K125A and R99A have reduced affinity for the 40S ribosome, which alleviates the translational repression of cellular transcripts. However, CoV-2 leader-containing transcripts instead become translationally repressed, perhaps due to a “nonproductive” interaction with nsp1 in the absence of proper engagement with residues R124/K125 or R99.

Article Snippet: pCDNA4TO-3XFLAG Halo delta 118-180 nsp1 , This paper , 175424 (Addgene).

Techniques: Preserving, Blocking Assay, Sequencing

Journal: Cell Reports

Article Title: The N-terminal domain of SARS-CoV-2 nsp1 plays key roles in suppression of cellular gene expression and preservation of viral gene expression

doi: 10.1016/j.celrep.2021.109841

Figure Lengend Snippet:

Article Snippet: pCDNA4TO-3XFLAG Halo delta 118-180 nsp1 , This paper , 175424 (Addgene).

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Transfection, Luciferase, SYBR Green Assay, Primer Extension Assay, Clone Assay, Software

Journal: bioRxiv

Article Title: Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma

doi: 10.1101/2024.03.05.583565

Figure Lengend Snippet: A) Graph showing expression of neural crest genes upregulated in OMM1.3-QR + UM DCCs as revealed from RNA-seq DEG. B) NR2F1 mRNA expression in OMM1.3 QR + /QR - FACS sorted cells isolated from the livers of mice after 3 months. C) Representative IF images of solitary or <8 cell cluster and >8 cells cluster of UM004 (left panel) and OMM1.3 (right panel) cells disseminated to the liver and stained for NR2F1 and HMB45. The graphs (lower panel) show the percentage of NR2F1 positive cells in each bin of cell cluster categories. Cells having strong HMB45 staining were used to quantify co-stained NR2F1. D) IF images (left) and quantification (right) for the expression of NR2F1 (green) in solitary or > 8 cell clusters of OMM1.3 cells cultured in 3D Matrigel. E) IF images showing NR2F1 expression (pink) in solitary DCCs or isolated small clusters (left column) or small UM cell clusters (middle column) at the metastasis-liver edge and in the tumor core (right column) in metastatic UM patient samples. The dotted squares in the panel are shown as a zoom-in view across the right side. Graphs represent quantification of NR2F1 expression level (mean fluorescent intensity, MFI) in the nucleus for the above-stained patients.

Article Snippet: Briefly, two separate NR2F1 gRNAs (guide 2, 5′-GAT​CCG​CAG​GAC​GAC​GTG​GC-3′; and guide 4, 5′-GGC​TGC​CGT​AGC​GCG​ACG​TG-3′) were cloned into pLentiCRISPRv2 (Addgene-52961).

Techniques: Expressing, RNA Sequencing Assay, Isolation, Staining, Cell Culture

Journal: bioRxiv

Article Title: Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma

doi: 10.1101/2024.03.05.583565

Figure Lengend Snippet: A) Graph showing expression of YAP1, TEAD’s, and YAP1 downstream target genes in OMM1.3 QR + cells as analyzed from RNA-seq data. B) YAP1 mRNA expression in OMM1.3 QR + (dormant) vs. QR - (proliferative) cells at 3 months. C ) IF images showing expression of YAP1 (red) in OMM1.3 solitary and >2 cell cluster cultured in 3D Matrigel. The graph shows quantification of nuclear MFI of YAP1 in solitary and >2 cells nuclei. D) Representative images of colonies formed by Dox-treated OMM1.3 cells transiently transfected with control or NR2F1-targeting siRNAs at day 7- and 14-days post-seeding, showing H2B-GFP and TdT expression. E) H2B-GFP label retention at day 7 and 14 in control and NR2F1 knockdown cells (Mann-Whitney test). F) Graph showing single cell and > 8-cluster cell colonies formed by control and NR2F1 knockdown OMM1.3 cells cultured on 3D Matrigel. G) Fold change in mRNA expression of indicated genes in OMM1.3 control or NR2F1 knockdown cells. H) Graph showing number of colonies (only cells > 8-cells were included in quantification) upon YAP1 KD. I) Representative IF images (left) and graphs (right) showing expression of YAP1 (green) and NR2F1 (red) in colonies formed by OMM1.3 control and YAP1 knockdown cells at day 7. J) Western blots showing expression of NR2F1, YAP1 and TEAD1 in uninduced (No Dox) and induced (Dox treated) UM004 cells. K) Graph showing colonies formed by uninduced and Dox-induced NR2F1 over-expressing UM004 cells after 7 days. L) Western blots showing expression of NR2F1 and YAP1 in UM004 control and NR2F1 KD cells. M) Graph showing events of colonies for control and NR2F1 KD UM004 cells at day 7 (n= 3). N) IF images of UM004 solitary and clusters DCCs showing expression of YAP1 post 3 months of UM004 cells dissemination to liver. Zoom-in images are shown in the same panel with marked boundaries indicating YAP1 protein expression. O) IF images showing expression of YAP1 and NR2F1 in solitary or small UM cell clusters in UM patient liver samples. Graphs show the quantification of the MFI of nuclear YAP1 and NR2F1 (arbitrary units A.U.) in solitary and big clusters (proliferative lesion) in UM patient liver samples.

Article Snippet: Briefly, two separate NR2F1 gRNAs (guide 2, 5′-GAT​CCG​CAG​GAC​GAC​GTG​GC-3′; and guide 4, 5′-GGC​TGC​CGT​AGC​GCG​ACG​TG-3′) were cloned into pLentiCRISPRv2 (Addgene-52961).

Techniques: Expressing, RNA Sequencing Assay, Cell Culture, Transfection, MANN-WHITNEY, Western Blot

Journal: bioRxiv

Article Title: Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma

doi: 10.1101/2024.03.05.583565

Figure Lengend Snippet: Western blots showing expression of indicated proteins in control and NR2F1 knockout OMM1.3 cells, and UM004 untreated or treated with dox to induce NR2F1 expression. A double asterisk indicates a non-specific band. Densitometric quantification (n=3) of YAP1 and TEAD1 expression levels in the OMM1.3 cell line is also shown. C&D) OMM1.3 and UM004 treated cells as in A processed for Immunoprecipitation (IP) with anti-TEAD1 antibodies followed by immunoblotting with anti-YAP1 antibodies. 10% of input and IgG antibodies were used as internal control and IgG was used as a negative control. E) Representative images of melanospheres (n=3) formed by control and CRISPR/cas9 NR2F1 KO OMM1.3 cells after 14 days in the organoid medium. Lower panel shows the quantification of average size of melanospheres in control and NR2F1 KO OMM1.3 cells. (Mann-Whitney test, p-value < 0.0001). F) Representative images of livers isolated 3 months after intra-splenic injection of OMM1.3 control and NR2F1-KO cells. The images presented here represent the extreme case of liver metastatic burden, and not all animals showed such massive growth. G) Images showing vimentin expression in mouse livers injected with OMM1.3 cells after 3 months to quantify metastatic burden (upper row). The middle row is the zoom-in view of the marked area, showing the morphology of disseminated UM cells in the liver. The lower row shows IF images of the same sections stained with HMB45 (green) antibody. The right panel shows the quantification of metastatic area calculated using vimentin-positive cells (p-value *<0.05 and **<0.02). H IF images showing expression of YAP1 in small UM cell clusters and proliferative lesions in mouse liver injected intra-splenically with OMM1.3 control and NR2F1-KO cells.

Article Snippet: Briefly, two separate NR2F1 gRNAs (guide 2, 5′-GAT​CCG​CAG​GAC​GAC​GTG​GC-3′; and guide 4, 5′-GGC​TGC​CGT​AGC​GCG​ACG​TG-3′) were cloned into pLentiCRISPRv2 (Addgene-52961).

Techniques: Western Blot, Expressing, Knock-Out, Immunoprecipitation, Negative Control, CRISPR, MANN-WHITNEY, Isolation, Injection, Staining

Journal: bioRxiv

Article Title: Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma

doi: 10.1101/2024.03.05.583565

Figure Lengend Snippet: A) Schematic showing primer pairs used for scanning the putative binding site of NR2F1 on YAP1 genomic locus (1000 bp upstream of TSS) B) NR2F1 ChIP-qPCR analysis in OMM1.3 cells shows the recruitment of NR2F1 upstream of the TSS of the YAP1 promoter. Isotype-matched IgG was used as negative antibody control. C&D) ChIP-qPCR showing enrichment of histone carrying activation marks (H3K4me3 & H3K27ac) and repression (H3K9me3) marks at the YAP1 gene using primer pair (-320bp) in OMM1.3 control and NR2F1 knockout cells, and UM004 dox-inducible NR2F1 cells. NR2F1 occupancy onto YAP1 genomic locus in UM004 cells was analyzed using primer pair (-320bp); n= 3, error bars = SD, p-values * < 0.05, ** < 0.02 and *** < 0.01. E) Volcano plot showing DEGs in OMM1.3 control and NR2F1 KO cells. F) Heatmap showing significantly upregulated and downregulated genes in OMM1.3 control and NR2F1 KO cells using RNA-seq data based on unbiased clustering and p-adj values. G&H) Distribution of biological pathways and processes predicted to be upregulated and downregulated as ranked by log 2 FC from the top 100 upregulated and downregulated genes in NR2F1 KO OMM1.3 cells compared to control OMM1.3 cells using the Enrichr program.

Article Snippet: Briefly, two separate NR2F1 gRNAs (guide 2, 5′-GAT​CCG​CAG​GAC​GAC​GTG​GC-3′; and guide 4, 5′-GGC​TGC​CGT​AGC​GCG​ACG​TG-3′) were cloned into pLentiCRISPRv2 (Addgene-52961).

Techniques: Binding Assay, Activation Assay, Knock-Out, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Lineage commitment pathways epigenetically oppose oncogenic Gαq/11-YAP signaling in dormant disseminated uveal melanoma

doi: 10.1101/2024.03.05.583565

Figure Lengend Snippet: Cut&Run analysis for H3K27me3 and H3K27ac marks was performed in control and NR2F1 KO OMM1.3 cell lines. Genomic regions with differences in signals were identified, and peaks with significant signal enrichment in either control or NR2F1 KO cells are shown as dot plots (right); CPM (average counts per million reads in a peak). C&D) Distribution of biological processes predicted to be modulated in NR2F1 KO cells based on differential peaks called for H3K27me3 mark as analyzed by Enrichr program. E) Distribution of H3K27me3 and H3K27ac peaks for YAP1 gene in control and NR2F1 KO condition (only one replicate is shown here). Tracks are normalized and scaled to the range by the integrative genomic viewer (IGV). F) Graph showing quantification of colonies formed by OMM1.3 cells grown on Matrigel after 14 days treated with DMSO and compound YM with different concentrations. G) Graph showing quantification of NR2F1 positive cells (< and > 8-cells cluster) following treatment with compound YM at different concentrations. H) Representative IF images showing expression of YAP1 (red) in colonies formed by OMM1.3 cells treated with 300nM of YM for 14 days and corresponding quantification of YAP1 positive cells (p-value < 0.05, students t-test ).

Article Snippet: Briefly, two separate NR2F1 gRNAs (guide 2, 5′-GAT​CCG​CAG​GAC​GAC​GTG​GC-3′; and guide 4, 5′-GGC​TGC​CGT​AGC​GCG​ACG​TG-3′) were cloned into pLentiCRISPRv2 (Addgene-52961).

Techniques: Expressing