Journal: bioRxiv

Article Title: Cell Type-Agnostic Optical Perturbation Screening Using Nuclear In-Situ Sequencing (NIS-Seq)

doi: 10.1101/2024.01.18.576210

Figure Lengend Snippet: (A) Outline of NIS-Seq reaction steps in comparison to previously established in-situ sequencing of barcoded mRNA . (B) NIS-Seq imaging results in comparison to cytosolic in-situ sequencing results obtained across three cell types. Scale bar, 50 μm. (C) Nuclei assignment between live cell imaging and NIS-Seq data across different objectives and timepoints. Nuclear staining patterns are mapped by two-dimensional FFT-accelerated high-pass filtered cross-correlation (top right panel). Overlay of nuclear signal reveals slight dislocation of nuclei between imaging timepoints (bottom left). Centers of gravity of CellPose-defined nuclei are assigned to nearest neighbors and ambiguous assignments are removed (bottom center and right). Assigned nuclei are color-coded by the same random color. Scale bar, 50 μm. (D) Raw images of 14 cycles of NIS-Seq barcode sequencing. Nuclear staining was performed at cycles 1, 4, 7, 10, and 13. Scale bar, 10 μm. (E) Quantitative spot intensities obtained from nuclei I and II highlighted in (D). Indicated on top is the base calling result, matching two members of the pooled lentiviral library used. (F) Fraction of nuclei mapping to known library member sequences. Unambiguous mapping is defined as more than two thirds of aggregated library-matching spot intensities within a nucleus mapping to a single library member. (G) Library coverage in transduced THP1 macrophages, measured by PCR-based NGS and NIS-Seq. Each library member covered in PCR-based sequencing is represented by one dot; dropping out sgRNAs, e.g., those targeting essential genes, are not shown. Jitter is added to values to visualize spot density even at low integer values. (H) Genome editing efficiencies of widely-used sgRNA-expressing lentivirus designs compared to constructs with an additional T7 promoter inserted for NIS-Seq in reverse-orientation. Genome editing efficiencies at three independent loci were assessed by NGS in HeLa-Cas9 cells transduced with indicated lentiviral constructs after four days of Puromycin selection and Cas9 induction.

Article Snippet: For THP1 screens, THP1-ASC-GFP-Cas9 CASP1/8 KO Brunello-iT7 cells were pre-differentiated overnight with 100 ng/ml PMA (Invivogen).

Techniques: Comparison, In Situ, Sequencing, Imaging, Live Cell Imaging, Staining, Expressing, Construct, Transduction, Selection

Journal: bioRxiv

Article Title: Cell Type-Agnostic Optical Perturbation Screening Using Nuclear In-Situ Sequencing (NIS-Seq)

doi: 10.1101/2024.01.18.576210

Figure Lengend Snippet: (A) Results of two replicates of genome-scale NIS-Seq perturbation screening in HeLa-Cas9-p65-mNeonGreen cells stimulated with IL-1β. Dots correspond to genes and axes indicate mean pixel-wise Pearson correlation between mNeonGreen and nuclear staining signals. (B) Collages of cellular images from (A) mapped to perturbed genes indicated. Shown is the mNeonGreen signal. (C) Arrayed hit validation in HeLa-Cas9-p65-mNeonGreen cells using alternative sgRNA sequences from the Toronto KO library v3. Top panel, exemplary mNeonGreen images of IL-1β-stimulated cells. Bottom panel, distribution of activation states, quantified by pixel-wise Pearson correlation between mNeonGreen and nuclear staining signals. Scale bar, 50 μm. (D) Results of two replicates of genome-scale NIS-Seq perturbation screening in HeLa-Cas9-p65-mNeonGreen cells stimulated with TNF-α. Dots correspond to genes, and axis positions indicate mean pixel-wise Pearson correlation between mNeonGreen and nuclear staining signals. (E) Collages of cellular images from (D) mapped to perturbed genes indicated. Shown is the mNeonGreen signal. (F) Arrayed hit validation in HeLa-Cas9-p65-mNeonGreen cells using alternative sgRNA sequences from the Toronto KO library v3. Top panel, exemplary mNeonGreen images of TNF-α-stimulated cells. Bottom panel, distribution of activation states, quantified by pixel-wise Pearson correlation between mNeonGreen and nuclear staining signals. Scale bar, 50 μm.

Article Snippet: For THP1 screens, THP1-ASC-GFP-Cas9 CASP1/8 KO Brunello-iT7 cells were pre-differentiated overnight with 100 ng/ml PMA (Invivogen).

Techniques: Staining, Activation Assay

Journal: bioRxiv

Article Title: Cell Type-Agnostic Optical Perturbation Screening Using Nuclear In-Situ Sequencing (NIS-Seq)

doi: 10.1101/2024.01.18.576210

Figure Lengend Snippet: (A) Results of two replicates of genome-scale NIS-Seq perturbation screening in THP1-Cas9-ASC-GFP-CASP1/8 DKO cells stimulated with Nigericin. Dots correspond to genes and axes indicate mean ratios of high-pass filtered GFP signal relative to the overall GFP signal per cell. (B) Collages of cellular images from (A) mapped to perturbed genes indicated. Shown are membrane stain (red) and ASC-GFP (green) signals. (C) Arrayed hit validation in THP1-Cas9-ASC-GFP cells using alternative sgRNA sequences from the Toronto KO library v3. Shown are fractions of cells with an ASC speck upon Nigericin stimulation in three replicate wells. (D) Cytokine secretion in response to two inflammasome triggers in wild type or clonal NLRP3-deficient THP1-ASC-GFP cells, measured by IL-1β ELISA. Shown are three replicate wells of stimulated cells.

Article Snippet: For THP1 screens, THP1-ASC-GFP-Cas9 CASP1/8 KO Brunello-iT7 cells were pre-differentiated overnight with 100 ng/ml PMA (Invivogen).

Techniques: Membrane, Staining, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: CRISPR-Cas and CRISPR-based screening system for precise gene editing and targeted cancer therapy

doi: 10.1186/s12967-024-05235-2

Figure Lengend Snippet: Schematic representative of CRISPR/Cas loci in Class 1 and Class 2 system. Class 1 system show multi-component effectors, while the Class 2 system have one effector. Three subgroups of Class 2 CRISPR systems are presented. Representative Type II-A CRISPR protein contains: Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9) and Streptococcus thermophilus Cas9 (StrCas9), all of which have the tracrRNA sequences. Type V CRISPR, which comprises Cas12a, Cas12b and Cas12c, exhibits distinct genome structures. Cas12b has the tracrRNA structure, while Cas12c only has one assistant protein cas1 for genome editing. Cas14 subgroup is not depicted in this figure. Type VI CRISPR systems show few assistant proteins to identify RNA virus, however, type VI-B has csx27 and csx28 proteins to regulate nuclease activity. Illustrated according to Ref [ , , , ].

Article Snippet: Cas9 , Addgene #73,179 , CAR-T cells Glioblastoma stem cells , CRISPRko , Gene dependencies , [ ] .

Techniques: CRISPR, Virus, Activity Assay

Journal: Journal of Translational Medicine

Article Title: CRISPR-Cas and CRISPR-based screening system for precise gene editing and targeted cancer therapy

doi: 10.1186/s12967-024-05235-2

Figure Lengend Snippet: Summary of Cas9 proteins and modified nCas9 and dCas9 genome editing tools. (A) PAM for SpCas9 is NGG, while PAM for SaCas9 is NNGRRT with the ability to cut DNA double helix. (B) Mutation of D10A leads to the formation of nCas9 while both mutations generate dCas9 protein. (C) nCas9 can be applied for base editing such as CBE and ABE, also for Base editor and developed as NICER to repair heterogenous mutation. (D) dCas9 was modified to generate CRISPRi, CRISPRa and CRISPR labeling tools. dCas9: dead Cas9. nCas9: nickase Cas9. CBE: Cytosine Base Editor, ABE: Adenine Base Editor. RT: reverse transcriptase. pegRNA: prime editing guide RNA.

Article Snippet: Cas9 , Addgene #73,179 , CAR-T cells Glioblastoma stem cells , CRISPRko , Gene dependencies , [ ] .

Techniques: Modification, Mutagenesis, CRISPR, Labeling, Reverse Transcription

Journal: Journal of Translational Medicine

Article Title: CRISPR-Cas and CRISPR-based screening system for precise gene editing and targeted cancer therapy

doi: 10.1186/s12967-024-05235-2

Figure Lengend Snippet: CRISPR screening ex vivo for cancer research

Article Snippet: Cas9 , Addgene #73,179 , CAR-T cells Glioblastoma stem cells , CRISPRko , Gene dependencies , [ ] .

Techniques: CRISPR, Ex Vivo, Biomarker Discovery, Ubiquitin Proteomics, Activation Assay, Plasmid Preparation, Drug discovery

Journal: Journal of Translational Medicine

Article Title: CRISPR-Cas and CRISPR-based screening system for precise gene editing and targeted cancer therapy

doi: 10.1186/s12967-024-05235-2

Figure Lengend Snippet: CRISPR screening in vivo for cancer therapy

Article Snippet: Cas9 , Addgene #73,179 , CAR-T cells Glioblastoma stem cells , CRISPRko , Gene dependencies , [ ] .

Techniques: CRISPR, In Vivo, Multiplex Assay, Genome Wide