Journal: bioRxiv

Article Title: A virally encoded high resolution screen of cytomegalovirus host dependencies

doi: 10.1101/2023.10.30.564793

Figure Lengend Snippet: a. The VECOS platform. A virus encoding an sgRNA in its genome infects a cell expressing Cas9. Upon infection, the sgRNA is expressed and interacts with Cas9 to induce site-specific mutations in the host genome. The viral genome replicates in the cells and produces new progeny containing the encoded sgRNA sequence. The level of viral genomes depends on the effect of the mutation induced by the virally encoded sgRNA. b. Examples of HCMV plaques in infected fibroblasts that are GFP positive (left) or GFP negative (right). c. Percentages of GFP positive and negative plaques formed by viruses budding from Cas9- expressing fibroblasts infected with HCMV encoding GFP and a sgRNA targeting GFP. Error bars represent the standard deviation of three biological replicates. d. Immunoblot analysis of RRM2 (top) and TRIP10 (bottom) protein expression at 3 dpi. Proteins were extracted from Cas9- or mCherry- (control) expressing fibroblasts infected with HCMV expressing either RRM2 or TRIP10 sgRNAs. GAPDH and UL44 were used as host and viral loading controls, respectively. e. Main steps in generating a VECOS library. Step I: A Custom Donor plasmid (pDONR-sgRNA) containing an sgRNA expression cassette (green) flanked by recombination sites (orange), and an HCMV BAC (HCMV-GW) containing the CcdB toxin gene (red) flanked by recombination sites (orange) and Chloramphenicol resistance gene (blue) were designed to facilitate introduction of a sgRNA library into the HCMV BAC via Gateway recombination. Step II: The Gateway recombination reaction products were transformed into optimized electrocompetent bacteria, CcdB toxin was used for negative selection (eliminating the parental BAC) and Chloramphenicol (Cm) was used for positive selection. Step III: The BAC library was introduced into fibroblasts via Adenofection to produce an infectious HCMV virus encoding the sgRNA.

Article Snippet: The Donor library or individual sgRNA plasmids were linearized using the NheI-HF enzyme (NEB) in a 50 µl reaction using 6 µg plasmid DNA and 1 µl enzyme, and the reaction was purified using Wizard SV Gel and PCR Clean-Up System (Promega).

Techniques: Virus, Expressing, Infection, Sequencing, Mutagenesis, Standard Deviation, Western Blot, Control, Plasmid Preparation, Transformation Assay, Bacteria, Selection

Journal: bioRxiv

Article Title: Heterotypic inter-GPCR ß-arrestin coupling regulates lymphatic endothelial junctional architecture in murine lymph nodes

doi: 10.1101/435776

Figure Lengend Snippet: ( A ) Schematic of S1PR1 modulator screening system Four lentiviral vectors were transduced into U2OS cell line to enable gene activation by SAM and monitoring S1PR1 activation by TANGO system. The cells introduced with SAM sgRNA library were starved with 0.5% charcoal treated FBS, then the Venus-positive population was sorted and next-gen sequence (NGS) analysis was carried out to identify the enriched SAM sgRNA sequences. ( B ) Scatter plot showing enrichment of sgRNAs after sorting. Most sgRNAs are equally distributed in the pre-sort sample (closed gray circles) while after sorting a small fraction of sgRNAs (2,770 out of 70,290 sgRNAs) were enriched and others were not detected (open blue circles). The y-axis shows the NGS reads of sgRNAs. ( C ) Identification of top candidate genes using the MAGeCK method . The names of top ten candidate genes are indicated.

Article Snippet: The single clones were isolated from antibiotics resistant cells by limiting dilution, then introduced with the SAM sgRNA library (a gift from Feng Zhang, Addgene #1000000057) at a low multiplicity of infection.

Techniques: Activation Assay, Sequencing

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Comparison of growth phenotypes for all elements between our pilot single-sgRNA library and Horlbeck et al. data, merged by gene name (n=20,228 elements). Growth phenotypes are reported as γ (log 2 fold-enrichment of T final over T 0 , per doubling) and correlated between experiments (r=0.82). ( B ) Comparison of growth phenotypes for all elements between our pilot dual-sgRNA library and Horlbeck et al. data, merged by gene name (n=20,228 elements). Growth phenotypes are reported as γ and correlated between experiments (r=0.83). ( C ) Comparison of growth phenotypes for all elements between our pilot single- and dual-sgRNA libraries, merged by gene name (n=21,239 with 20,228 targeting elements and 1011 non-targeting elements). Growth phenotypes are reported as γ and correlated between experiments (r=0.86). ( D ) Comparison of true and false-positive rates in single element screens. ‘Positives’ (n=1363 elements) were defined as genes with a K562 CRISPRi growth screen p-value <0.001 and γ<–0.05 , and ‘negatives’ were defined as non-targeting control sgRNA pairs (n=1011 elements). ( E ) Comparison of recombination rates for non-targeting dual-sgRNA elements between replicates of our K562 growth screen. Non-targeting elements with a growth phenotype (γ>0.05 or γ<−0.05) were excluded (n=973 elements). Recombination rates were weakly correlated between replicates (r=0.30). ( f ) Comparison of recombination rates for all dual-sgRNA elements between replicates of our K562 growth screen (n=20,387 elements). Recombination rates were strongly correlated between replicates (r=0.77). ( G ) Comparison of recombination rates and growth phenotypes for all dual-sgRNA elements in our K562 growth screen (n=20,387 elements). Growth phenotypes are reported as γ. Recombination rates were strongly anticorrelated with growth phenotypes (r=−0.84).

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Comparison, Control

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Schematics of CRISPRi transcription repressor domains and general lentiviral expression construct used for all CRISPRi effectors. UCOE = ubiquitous chromatin opening element; SFFV = spleen focus-forming virus promoter; P2A = ribosomal skipping sequence; WPRE = woodchuck hepatitis virus post-transcriptional regulatory element. Further information on repressor domains and lentiviral expression constructs can be found in the main text and Materials and methods. ( B ) Experimental design to test effects of stable expression of each CRISPRi effector on growth and transcription in K562 cells. ( C ) Growth defects of effector-expressing cells, measured as the log 2 of the ratio of mCherry-negative (effector-expressing) to mCherry-positive (not effector-expressing) cells in each well normalized to the same ratio on day 0. mCherry levels were measured for 19 days after pooling cells. Data represent mean ± SD from three independent transductions of expression constructs. p-Values are from an unpaired two-tailed t-test comparing D19 values for each sample to the D19 value for the ‘no plasmid’ sample. Average percent growth defect per day is the log 2 D19 value divided by the number of days, multiplied by 100 for a percent value. ( D ) Clustered heatmap of correlation of transcript counts from K562 cells expressing indicated CRISPRi effectors or a GFP control. Correlations across samples were calculated using normalized counts (reads per million) for all genes with mean normalized count >1 and then clustered using the Ward variance minimization algorithm implemented in scipy. r 2 is squared Pearson correlation. Data represent three independent transductions of expression constructs. ( E ) Number of differentially expressed genes ( p <0.05) for cells expressing each effector versus cells expressing GFP only. p -Values were calculated using a Wald test and corrected for multiple hypothesis testing as implemented in DeSeq2. Figure 2—source data 1. p-Values and growth defects depicted in . Figure 2—source data 2. Data depicted in .

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Expressing, Construct, Virus, Sequencing, Two Tailed Test, Plasmid Preparation, Control

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: Design of constructs for CRISPR interference (CRISPRi) effector expression.

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Construct, CRISPR, Expressing

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Experimental design to measure knockdown mediated by different CRISPR interference (CRISPRi) effectors by delivering single guide RNAs (sgRNAs) targeting either essential genes or cell surface markers. ( B ) Depletion of K562 cells expressing essential gene-targeting sgRNAs and different CRISPRi effectors, measured as the ratio of mCherry-positive (sgRNA-expressing) to mCherry-negative (not sgRNA-expressing) cells in a given well. mCherry levels were measured for 12 days after transduction, starting on day 3. Data from two replicate transductions. ( C ) Percent knockdown of cell surface markers by different CRISPRi effectors in K562 cells. Cell surface marker levels were measured on day 6 post-transduction by staining with an APC-conjugated antibody. Knockdown was calculated as the ratio of median APC signal in sgRNA-expressing cells and median APC signal in cells expressing a non-targeting control sgRNA after subtraction of background APC signal. Data from two replicate transductions. Cells expressing dCas9 and a strong CD55-targeting sgRNA are represented by a single replicate. ( D ) Distribution of anti-CD151 signal intensity (APC) in individual cells from one representative transduction. Data from second replicate are shown in . Knockdown was quantified as in C as the ratio of the median APC signals. ( E ) Percentage of cells without observable knockdown despite expressing a strong sgRNA, as quantified from the fluorescence distributions.

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Knockdown, CRISPR, Expressing, Transduction, Marker, Staining, Control, Fluorescence

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Depletion of K562 cells expressing essential gene-targeting single guide RNAs (sgRNAs) and different CRISPRi effectors, measured as the ratio of mCherry-positive (sgRNA-expressing) to mCherry-negative (not sgRNA-expressing) cells in a given well, as in . mCherry levels were measured for 12 days after transduction, starting on day 3. Data from two replicate transductions. ( B ) Distribution of anti-CD151 signal intensity (APC) in K562 cells expressing indicated CRISPRi effectors from second replicate transduction. Knockdown was quantified as in . ( C ) Distribution of anti-CD81 signal intensity (APC) in K562 cells expressing indicated CRISPRi effectors from two replicate transductions. Knockdown was quantified as in . ( D ) Distribution of anti-CD55 signal intensity (APC) in K562 cells expressing indicated CRISPRi effectors from two replicate transductions. Cells expressing dCas9 and the CD55-targeting sgRNA are represented by a single replicate. Knockdown was quantified as in .

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Expressing, Transduction, Knockdown

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet: ( A ) Distribution of anti-B2M signal intensity (APC) in individual RPE1 (left) and Jurkat (right) cells expressing indicated CRISPR interference (CRISPRi) effectors and single guide RNAs (sgRNAs). Knockdown was calculated as the ratio of median APC signal in transduced (sgRNA-expressing) cells and median APC signal in non-transduced cells in the same well, after subtraction of background APC signal. ( B ) Depletion of indicated cell surface markers in HepG2 (top), HuTu-80 (middle), and HT29 (bottom) cells expressing Zim3-dCas9. Cell surface marker levels were measured 6–14 days post-transduction by staining with APC-conjugated antibodies. Knockdown was calculated as the ratio of median APC signal in sgRNA-expressing cells and median APC signal in cells expressing a non-targeting control sgRNA after subtraction of background APC signal. ( C ) Distribution of anti-B2M signal intensity (APC) in individual K562 cells expressing indicated CRISPRi effectors and sgRNAs. The Zim3-dCas9 (Hygro) cell line was generated by transduction followed by hygromycin selection and does not express a fluorescent protein. Knockdown was calculated as in A .

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Expressing, CRISPR, Knockdown, Marker, Transduction, Staining, Control, Generated, Selection

Journal: eLife

Article Title: Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors

doi: 10.7554/eLife.81856

Figure Lengend Snippet:

Article Snippet: The human CRISPRi dual-sgRNA libraries with IBCs are available from Addgene (hCRISPRi_dual_1_2, Addgene #187246; hCRISPRi_dual_3_4, Addgene #187247; hCRISPRi_dual_5_6, Addgene #187248).

Techniques: Stable Transfection, Marker, Flow Cytometry, Recombinant, Plasmid Preparation, Sequencing, Expressing, Purification, Amplification, Transfection, Software, Genome Wide

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: (A) Summary of gene deletion vs. gene overexpression approaches. (B) Rationale for the present study. (C) Experimental outline of the present study. (D) HEK293 and Jurkat cells were inoculated with different volumes of Ebola or rabies pseudovirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This revealed that Jurkat cells are largely refractory to Ebola or rabies pseudovirus entry, relative to HEK293 cells. (E) Construction of a clonal Jurkat cell line, known as “Jurkat C6”, stably expressing a degron-tagged CRISPRa construct ( left ). Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence or absence of TMP (1 μM) for 3 days, followed by flow cytometry to detect hCD19 expression ( right ). (F) At each of the indicated points of the genome-wide CRISPRa screen, the cell population was challenged with Ebola or rabies pseudovirus, and cell infectivity was evaluated by flow cytometry. Upon successive rounds of the screen, the cell population became progressively more susceptible to infection to either Ebola or rabies pseudovirus, respectively. (G) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for Ebola pseudovirus entry. (H) sgRNA distribution upon successive rounds of the genome-wide CRISPRa screen for rabies pseudovirus entry.

Article Snippet: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

Techniques: Over Expression, Marker, Mutagenesis, Flow Cytometry, Infection, Stable Transfection, Expressing, Construct, Transduction, Genome Wide

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) Plasmids used to pseudotype non-replicating lentiviruses with either Ebola or rabies envelope proteins. EBOV-GP: glycoprotein of Ebola virus, Makona variant. RABV-GP N2C: glycoprotein of rabies virus, N2C variant. B) HEK293 and Jurkat cells were inoculated with different volumes of VSV envelope protein-pseudotyped lentivirus encoding a cell-surface marker (mCD19t; a truncated mutant of mouse CD19), followed by flow cytometry to determine the percentage of infected cells. This served as a positive control to confirm that Jurkat cells and HEK293 cells are both susceptible to VSV pseudovirus entry. C) Jurkat C6 cells were transduced with sgRNA targeting the endogenous human CD19 gene, in the presence of different TMP concentrations (0-4 μM) for 2-3 days, followed by flow cytometry to detect human CD19 expression.

Article Snippet: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

Techniques: Virus, Variant Assay, Marker, Mutagenesis, Flow Cytometry, Infection, Positive Control, Transduction, Expressing

Journal: bioRxiv

Article Title: Elucidating genes sufficient for viral entry into cells through sequential genome-wide CRISPR activation screens

doi: 10.64898/2026.03.06.710083

Figure Lengend Snippet: A) NGFR was expressed in Jurkat C6 cells using CRISPRa, or alternatively, Jurkat cells using cDNA expression. NGFR -expressing or control cells were then inoculated with rabies pseudovirus encoding mCD19t. Flow cytometry was then performed to determine the percentage of infected cells. This revealed that NGFR expression significantly increased the susceptibility of Jurkat cells to rabies pseudovirus infection. As positive controls, flow cytometry was used to confirm successful delivery of the sgRNA construct as part of the CRISPRa workflow (as denoted by BFP expression) and that NGFR was expressed (upon cDNA expression). B) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression. L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with rabies pseudovirus encoding hEGFRt (a truncated mutant of human EGFR). Flow cytometry was then performed to determine the percentage of infected cells. This revealed that L-SIGN or DC-SIGN expression significantly increased the susceptibility of Jurkat cells and primary T cells to Ebola pseudovirus infection. Cells expressing the highest levels of L-SIGN and DC-SIGN were preferentially infected by Ebola pseudovirus. C) L-SIGN or DC-SIGN were expressed in primary human CD4 + T cells using cDNA expression, and then L-SIGN -expressing, DC-SIGN -expressing, or control cells were inoculated with GFP -expressing Ebola virus or zsGreen -expressing Sudan virus under BSL4 containment. On days 0, 1, and 2 post-infection, flow cytometry was performed to determine the percentage of infected cells and qPCR was performed on cell culture supernatants to quantify viral genome replication. This revealed that L-SIGN or DC-SIGN expression enabled authentic Ebola and Sudan virus entry into primary human T cells, but viral genome replication was impaired, perhaps reflective of cell-intrinsic restriction factors.

Article Snippet: To achieve CRISPRa-mediated overexpression, sgRNA sequences were cloned into the lentiviral sgRNA library backbone plasmid (Addgene, 84832) using the restriction sites BstX1 and BlpI.

Techniques: Expressing, Control, Flow Cytometry, Infection, Construct, Mutagenesis, Virus, Cell Culture