Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: Mouse embryonic fibroblasts (MEF) stably expressing Flag-Cas9-EGFP were transduced with a lentiviral CRISPR-library. Cells were treated three times with PMT(C1165S)DTa and surviving cells grown. Genomic DNA was extracted, inserted sgRNA amplified and sequenced.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Stable Transfection, Expressing, Transduction, CRISPR, Amplification

Journal: bioRxiv

Article Title: Genome wide CRISPR screen for Pasteurella multocida toxin (PMT) binding proteins reveals LDL Receptor Related Protein1 (LRP1) as crucial cellular receptor

doi: 10.1101/2022.08.04.502755

Figure Lengend Snippet: The volcano plot shows gene expression changes between cells treated with lethal PMT-DTa chimera and those only transduced with the CRISPR library. All reads were mapped locally using BWA-MEM [ ,6], then quantified with featureCounts [4], and finally fold changes between the condition were calculated by DESeq2 [7] (see galaxy history). The volcano plot was drawn with the bioinfokit toolkit [8]. Significantly enriched genes, having a positive fold change above 0.584 and a p-value lower or equal 5%, are shown in blue. The significance thresholds are marked by gray-dotted lines. The ten most significant genes are highlighted with their name. Non-significant genes are colored in gray.

Article Snippet: We utilized a well-functioning and validate genome-wide mouse lentiviral CRISPR guide RNA library v2 (Addgene).

Techniques: Gene Expression, Transduction, CRISPR

Journal: bioRxiv

Article Title: Genome scale CRISPR screens identify actin capping proteins as key modulators of therapeutic responses to radiation and immunotherapy

doi: 10.1101/2024.01.14.575614

Figure Lengend Snippet: a, Schematic of the B16F10 genome-scale CRISPR KO screen is shown on the left. Validation experiment of WT B16F10 and Capza3 KO B16F10 cell survival following co-culture with OT-I CD8+ T cells is shown on the right. b, Screen analysis plot (left) and top 6 gRNAs enriched (right) in surviving B16F10 cells after low-dose radiation (1 Gy) treatment and co-culture with CD8 cytotoxic T cells. Results shown are from 2 biologic repeats of the screen. c, Screen analysis plot (left) and top 6 gRNAs enriched (right) in surviving B16F10 cells after low-dose radiation (1 Gy) treatment and anti-PD1 antibody treatment during co-culture with CD8 cytotoxic T cells. Results shown are from 2 biologic repeats of the screen. d, Screen analysis plot (left) and top 6 gRNAs enriched (right) in surviving B16F10 cells after anti-PD1 antibody treatment during co-culture with CD8 cytotoxic T cells. Results shown are from 2 biologic repeats of the screen. e, Validation experiment of WT B16F10 and Capza3 KO B16F10 cell survival. Capza3 KO B16F10 cells were transduced with lentivirus containing the Capza3 targeting gRNA and a GFP reporter. Prior to transduction of B16F10 cells, some cells were isolated to be used as a WT control. Flow analysis of % GFP positive cells (B16F10 Cas9 cells transduced with lentivirus that contains the Capza3 -targeting gRNA) following either: 1) co-culture with CD8 cytotoxic T cells or 2) treatment with low-dose radiation (1 Gy) and anti-PD1 antibody during co-culture with CD8 T cells. Representative plots are shown on the left and quantification is shown on the right. Significance testing was performed with two-way ANOVA. Validation experiments were performed using three biological replicates for each treatment condition. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Brie genome-scale CRISPR library , Addgene , 73633 , CRISPR lentiviral library.

Techniques: CRISPR, Biomarker Discovery, Co-Culture Assay, Transduction, Isolation, Control

Journal: bioRxiv

Article Title: Genome scale CRISPR screens identify actin capping proteins as key modulators of therapeutic responses to radiation and immunotherapy

doi: 10.1101/2024.01.14.575614

Figure Lengend Snippet:

Article Snippet: Brie genome-scale CRISPR library , Addgene , 73633 , CRISPR lentiviral library.

Techniques: CRISPR, Luciferase, Transfection, Control, Plasmid Preparation

Journal: Cell Reports Methods

Article Title: A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

doi: 10.1016/j.crmeth.2023.100463

Figure Lengend Snippet: Study work flow and design (A) Work flow steps from clinical specimens to reporting. (B) CRISPRclean workflow. (C) Data analysis work flow to (i) estimate rRNA composition, (ii) report taxonomic classification and the abundance of pathogens and co-infections as percentage of microbiome (non-human) reads, (iii) calculate pathogen genome coverage metrics, (iv) report antimicrobial resistance (AMR), and (v) investigate host gene expression.

Article Snippet: For site A specimens, 10ng of each RNA sample was used as input for library preparation using the CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion kit (Jumpcode Genomics), which targets human and bacterial rRNA for depletion.

Techniques: Gene Expression

Journal: Cell Reports Methods

Article Title: A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

doi: 10.1016/j.crmeth.2023.100463

Figure Lengend Snippet: Performance characteristics of CRISPRclean technology Five and 50 ng of total RNA from a human fecal reference sample was used as input into the CRISPRclean Plus or Illumina RiboZero Plus workflows. (A) Ribosomal RNA alignment. The percentage of reads aligning to rRNA species was calculated for non-depleted (No Depl) and depleted (Depl) sample libraries at the specified RNA input along with standard error bars. Depletion was performed with either Illumina RiboZero Plus (ILMN, purple bars) or CRISPRclean Plus (CRISPRclean, blue bars). Results show that CRISPRclean Plus is more effective at removing rRNA from metatranscriptomic libraries than Illumina RiboZero Plus. Data are represented as the mean of triplicates ±SD. (B) Number of bacterial species. Sequencing data from non-depleted (No Depl) or depleted (Depl) libraries treated with Illumina RiboZero Plus or CRISPRclean Plus were processed using the Kraken2-Bracken work flow and used to calculate the number of bacterial species with RPM ≥ 10. CRISPRclean Plus enabled the discovery of more bacterial species than Illumina RiboZero Plus at both RNA inputs. (C and D) High correlation of bacterial counts between non-depleted and depleted control samples at 5 ng (C) and 50 ng (D). Libraries were generated from fecal reference control RNA and subjected to a protocol with and without CRISPRclean Plus depletion. Sequencing reads from both treatments were processed using the Kraken2-Bracken work flow and the resulting bacterial species read counts from depleted libraries (y axis) were compared with non-depleted libraries (x axis). Bacterial species counts from CRISPRclean-depleted libraries showed high correlation with species counts from non-depleted libraries at both 5- and 50-ng RNA inputs ( r 2 = 0.898, left panel; r 2 = 0.9944, right panel, respectively) confirming that CRISPRclean treatment produces little bias with respect to bacterial species identification and read count measurement. Average of triplicates was used for the regression analysis.

Article Snippet: For site A specimens, 10ng of each RNA sample was used as input for library preparation using the CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion kit (Jumpcode Genomics), which targets human and bacterial rRNA for depletion.

Techniques: Sequencing, Control, Generated

Journal: Cell Reports Methods

Article Title: A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

doi: 10.1016/j.crmeth.2023.100463

Figure Lengend Snippet: Ribosomal RNA composition before and after CRISPRclean depletion of site A specimens (A) The percentage of reads from all site A libraries (n = 180) that align to different species of RNA is shown before and after depletion with CRISPRclean Plus. Following depletion, an increase in aligned reads for eukaryotic non-rRNA (dark blue), bacterial non-rRNA (blue), and viruses (light blue) is observed. A decrease in aligned reads is observed for eukaryotic rRNA (pink) and bacterial rRNA (purple). (B) The percentage of rRNA-aligned reads, calculated as an average of all libraries from site A (n = 180), was determined for bacteria (blue) and eukaryotes (purple). The percentage of aligned reads is shown with and without CRISPRclean depletion. CRISPRclean depletion removes nearly all bacterial and eukaryotic RNA. (C and D) The distribution of percent of aligned reads using Kraken2 is shown before (C) and after depletion (D) with CRISPRclean Plus. Prior to depletion, the majority of read alignments consist of eukaryotic rRNA (gray box) and eukaryotic non-rRNA (blue box; note: additional values outside the mean and its confidence limits are provided by the dots); however, after depletion, a large relative increase is observed of reads aligning to eukaryotic non-rRNA and bacterial non-rRNA (yellow box).

Article Snippet: For site A specimens, 10ng of each RNA sample was used as input for library preparation using the CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion kit (Jumpcode Genomics), which targets human and bacterial rRNA for depletion.

Techniques: Bacteria

Journal: Cell Reports Methods

Article Title: A CRISPR-enhanced metagenomic NGS test to improve pandemic preparedness

doi: 10.1016/j.crmeth.2023.100463

Figure Lengend Snippet:

Article Snippet: For site A specimens, 10ng of each RNA sample was used as input for library preparation using the CRISPRclean Plus Stranded Total RNA Prep with rRNA Depletion kit (Jumpcode Genomics), which targets human and bacterial rRNA for depletion.

Techniques: Virus, Illumina Sequencing, Software

Journal: Communications Biology

Article Title: The repurposing of type I-E CRISPR-Cascade for gene activation in plants

doi: 10.1038/s42003-019-0637-6

Figure Lengend Snippet: Engineering type I-E Cascade complex formation in Zea mays . a Native type I-E CRISPR-Cas locus from S. thermophilus DGCC7710. CRISPR repeats are indicated with a black diamond and the spacer region is in blue. CasE cleaves the primary CRISPR RNA (crRNA) transcript after the hairpin in the CRISPR repeat yielding the mature crRNA depicted. The Cascade complex guided by the crRNA in the vicinity of a 5′ PAM recognizes a double stranded DNA target as illustrated. b Zea mays optimized Cascade and crRNA expression constructs resulting in Cascade complex formation and gene activation. Zm Zea mays, UBI ubiquitin, NLS nuclear localization signal, PinII potato proteinase inhibitor terminator, CBF1 sequence encoding C-terminal acidic transcriptional activation domain from Arabidopsis cold binding factor 1

Article Snippet: J.K.Y., S.L.G. and R.B. have filed patent applications related to genome engineering with type I CRISPR systems, J.K.Y., S.L.G., S.J., L.W., P.N., and B.V. are employees of Corteva Agriscience, and R.B. is a co-founder of Locus Biosciences and Intellia Therapeutics, and a shareholder of Caribou Biosciences, Inari and Invaio.

Techniques: CRISPR, Expressing, Construct, Activation Assay, Sequencing, Binding Assay