Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet: Technical performance and quality control of in situ sequencing by synthesis (SBS). Data are from a screen in A549 cells with a CROPseq-puro library of 5,738 sgRNAs43. (a) Example compensation matrix used for correcting spectral cross-talk between SBS imaging channels. (b) Spectral compensation of the two-channel combinations with the most cross-talk (T vs G and C vs A) at the first and last cycle of an SBS experiment. Mapped reads are those with barcode sequences exactly matching expected sequences from the designed sgRNA library. Dotted lines in the compensated plots demarcate the decision boundary for base calling. (c) Plotting read mapping rate and mapped reads per cell against increasing thresholds on the peak parameter demonstrate that most non-mapping reads are excluded by thresholding this value. (d) Longer read lengths provide increased confidence of mapped reads representing true sequencing spots from barcode mRNA. Plotting plate heatmaps of quality control metrics such as read mapping rate (e), total cells imaged (f), and fraction of cells with reads mapping to one expected barcode sequence (g) is useful for evaluating the quality of an experiment and identifying potential issues.

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: In Situ, Sequencing, Imaging

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet: Overview of optical pooled screening. (a) Experimental workflow. First, a pooled sgRNA library is designed, packaged into lentivirus, and delivered into Cas9-expressing cells. A live-cell or fixed-cell imaging assay is performed to generate an optical phenotypic profile of individual cells. The sgRNA spacer sequences in each cell are then amplified and read out by in situ sequencing by synthesis (SBS), consisting of cycles of dye incorporation, imaging, and cleavage. Finally, sgRNA-encoded perturbations are mapped to phenotypic scores at the single-cell level, with candidate genes identified through various statistical approaches. (b) Schematic of the in situ SBS process. The sgRNA is expressed as a polyadenylated mRNA transcript from an integrated copy of the CROPseq vector. After fixation and permeabilization, a locked nucleic acid (LNA)-modified primer is used to reverse transcribe a cDNA copy of the sgRNA sequence. After glutaraldehyde and formaldehyde post-fixation, the mRNA is digested and a padlock probe is hybridized to cDNA regions flanking the sgRNA sequence. The padlock probe is then extended and ligated to copy the sgRNA sequence into a single-stranded circularized DNA. This circularized DNA serves as a template for rolling circle amplification with Phi29 polymerase, the amplified product of which contains tandem repeats of the sgRNA spacer sequence. These sequences are read out by successive cycles of SBS.

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: Expressing, Imaging, Amplification, In Situ, Sequencing, Plasmid Preparation, Modification

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet: Troubleshooting table

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: Transformation Assay, Electroporation, Plasmid Preparation, Amplification, Transfection, Transduction, Fluorescence, Imaging, Flow Cytometry, Expressing, Transferring, In Situ, Sequencing, Evaporation, Incubation, Buffer Exchange, Staining, Diffusion-based Assay, Selection, Infection, Concentration Assay, Microscopy, Software

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet:

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: Concentration Assay, Plasmid Preparation

Journal: Nature protocols

Article Title: Pooled genetic perturbation screens with image-based phenotypes

doi: 10.1038/s41596-021-00653-8

Figure Lengend Snippet:

Article Snippet: NGS validation primers for CROPseq-puro vector (Integrated DNA technologies) NGS_CROPseq-puro_P5: ACACGACGCTCTTCCGATCTtcttgtggaaaggacgaaacNGS_CROPseq-puro_P7: CTGGAGTTCAGACGTGTGCTCTTCCGATCTaagcaccgactcggtgccac.

Techniques: Concentration Assay

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: A modified CROP-seq sgRNA lentiviral expression vector (pJR107) was derived from the parental vector pBA950 ( https://www.addgene.org/122239/ ) by incorporating a GFP fluorescent marker and a UCOE element upstream of the EF1alpha promoter to prevent marker silencing. sgRNA targeting sequences were appended with flanking sequence, BstX1/BlpI overhangs, and PCR adapters.

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:

Article Snippet: A modified CROP-seq sgRNA lentiviral expression vector (pJR107) was derived from the parental vector pBA950 ( https://www.addgene.org/122239/ ) by incorporating a GFP fluorescent marker and a UCOE element upstream of the EF1alpha promoter to prevent marker silencing. sgRNA targeting sequences were appended with flanking sequence, BstX1/BlpI overhangs, and PCR adapters.

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