Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: Empirical design of the HD CRISPR library. Schematic illustration of the HD CRISPR library design process. sgRNA sequences that were previously used in negative selection screens contained in the GenomeCRISPR database were annotated with information on rationally selected sequence and phenotype features. All negative selection screens were reanalyzed with BAGEL. High-quality experiments were selected based on how well reference core and nonessential gene sets could be separated in those screens. sgRNAs with high on-target activity were then determined as sequences that both target an essential gene (as determined by BAGEL) and that rank among the 20% most strongly depleted sequences in the screen. Next, sgRNAs that showed unexpected phenotypes compared to other sgRNAs targeting the same gene were flagged as outliers with potential off-target effects. sgRNA sequences were then selected from the resulting pool of sequences to design a genome-wide library consisting of two mutually exclusive sub-libraries A and B, prioritizing sgRNAs with high on-target and low off-target activity

Article Snippet: Oligos encoding the HD CRISPR library A and B sgRNA sequences and flanking regions were ordered as an oligo pool from Twist Biosciences.

Techniques: CRISPR, Selection, Sequencing, Activity Assay, Genome Wide

Journal: BMC Biology

Article Title: Genome-scale CRISPR screening at high sensitivity with an empirically designed sgRNA library

doi: 10.1186/s12915-020-00905-1

Figure Lengend Snippet: Empirically selected sgRNAs in the HD CRISPR library. a Distribution of log2 fold changes for sgRNAs targeting core essential reference genes. The blue curve represents sequences with strong on-target phenotypes. The red curve represents sgRNAs with weak on-target phenotypes (see “ ”). b Phenotypes of sgRNAs targeting the core essential gene NOP2 for 4 screens performed with the library described in Wang et al. . Compared to other NOP2-targeting sgRNAs, sgRNA 9 showed unexpectedly weak depletion in these experiments and was thus labeled “ineffective”. c Distribution of log2 fold changes for sgRNAs targeting nonessential reference genes. The blue curve represents sequences with low off-target activity. The red curve represents sgRNAs that led to unexpectedly strong phenotypes. d Phenotypes of sgRNAs targeting the nonessential gene KRT35 in 4 screens performed with the library described in Wang et al. . Unlike other KRT35-targeting sgRNAs, sgRNA 8 consistently displayed toxic phenotypes in these experiments and was therefore marked as “toxic”. e Percentage of sgRNA sequences in sub-libraries A (left) and B (right) that could be selected based on empirical evidence from published screening experiments. Empirical essential sgRNAs were selected based on inducing a viability phenotype, empirical nonessential sgRNAs based on the absence of a toxic phenotype. f – h Calculated sequence scores applying either the rule set 2 , the DeepHF or the Hart et al. algorithms. Score performance of the HD CRISPR sub-libraries A and B was benchmarked against the libraries whose design is based on respective scores (Brunello for rule set 2, TKOv3 for Hart et al.) if available as well as the GeCKOv2 library and a random sample of sgRNAs from published libraries. The DeepHF score was used as an independent measure none of the investigated libraries was designed on. i – k Comparison of sgRNA scores for empirically and de novo-designed sgRNAs within the HD CRISPR sub-libraries

Article Snippet: Oligos encoding the HD CRISPR library A and B sgRNA sequences and flanking regions were ordered as an oligo pool from Twist Biosciences.

Techniques: CRISPR, Labeling, Activity Assay, Sequencing

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: VSV-eGFP possessed a sensitivity to type I IFN-triggered antiviral response. (A) Treatment with exogenous type I IFNs induces the expression of hundreds of ISGs, allowing the establishment of a so-called antiviral state against pathogen invasion in host cells. (B) Representative Flow cytometry analysis of EGFP expression rate in VSV-eGFP-infected IBRS-2 cells mock-treated (top), treated with RUX (500 nM) (middle, upper) and IFN (10 ng/mL) (middle, lower) alone or in combination (bottom). Cells without infection were tested as a background fluorescence intensity control (not shown in the histogram). VSV-eGFP replication was effectively inhibited by type I IFN treatment but subsequently restored by supplementation of RUX. (C) Graphs showing Flow cytometry data analysis indicating the reversion of IFN inhibition by addition of RUX. Data are expressed as mean ± SEM. ***P < 0.001.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: Expressing, Flow Cytometry, Infection, Fluorescence, Control, Inhibition

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: Identification of ISGs induced by exogenous type I IFNs using RNA-Seq. (A) Differentially expressed genes ( p value < 0.05, twofold or more change and FPKM value greater than 1 in at least one sample) for each IFN-treated sample are depicted numerically. (B) Correlation matrix of all 5 IFN-treated samples (based on Pearson correlation coefficients). (C) Upset plot of up-regulated DEGs in IFN-treated samples. Of note, all the up-regulated DEGs in the blue bars are clustered into ISG family.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: RNA Sequencing

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: Expression levels of select ISGs in type I IFN-treated IBRS-2 cells. The font size of each ISG is directly proportional to its average fold change in type I IFN-treated IBRS-2 cells normalized to mock-treated cells. Of note, ISGs with biggest font size demonstrated highest expression levels with fold changes greater than 1000.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: ISG-targeting CRISPR screen identifies a subset of genes as potential key effectors of the IFN response to VSV-eGFP replication. (A) Schematic of ISG-targeting lentiCRISPR screen to identify antiviral effectors mediating IFN-α 2a-induced antiviral response to VSV-eGFP. (B) Amplification of sgRNA expression cassettes in eGFP-positive IBRS-2 knockout cells. (C) Bar plots show the top 25 most enriched hits in the context of IFN-α 1b, IFN-α 2a and IFN-β. (D) Overlap between the top 25 most enriched ISGs in the context of IFN-α 1b, IFN-α 2a and IFN-β.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: CRISPR, Amplification, Expressing, Knock-Out

Journal: Frontiers in Immunology

Article Title: Establishment of a CRISPR/Cas9 knockout library for screening type I interferon-inducible antiviral effectors in pig cells

doi: 10.3389/fimmu.2022.1016545

Figure Lengend Snippet: The effects of top four ISGs on VSV-eGFP replication. (A) Schematic representation of the gateway-compatible bicistronic lentiviral vectors used to stably overexpress ISGs. The viral backbone carries the ISG-IRES-TagRFP overexpression cassette under the CMV promoter. In parallel, control vector GFP-IRES-TagRFP was also designed. (B) Fluorescent micrographs of IBRS-2 cells in culture 24 h after transduction with the control vector GFP/TagRFP. (C) Fluorescent micrographs of IBRS-2 cells in culture 24 h after transduction with ISG/TagRFP vectors. (D) Schematic demonstration of workflow of transduction and virus infections. (E) The TCID 50 titration of VSV-eGFP titers in the vector control and ISG/TagRFP overexpression IBRS-2 cells. The experiment was repeated three times with replicate each. **P < 0.01.

Article Snippet: Oligos encoding the sgRNA library with ~1908 specific sgRNA sequences targeting 359 ISGs were synthesized by a programmable microarray using the Synthesizer (GenScript, Wuhan) and cloned as a pool into lentiCRISPRv2 vector.

Techniques: Stable Transfection, Over Expression, Control, Plasmid Preparation, Transduction, Virus, Titration