knockout toxoplasma Search Results


crispr library  (Addgene inc)
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Addgene inc crispr library
(A) Schematic representation of sub-library <t>CRISPR</t> screens both in vitro and in vivo (mouse). Guide libraries targeting 66 genes (including controls) of 5 guides per gene, were constructed. For in vitro screen; the guide library was transfected into ME49:Cas9 reporter line, which expresses RFP constitutively using the Gra1 promoter ( gra1 promoter -RFP)and mNeonGreen (mNG) under control of the bradyzoite-specific BAG1 promoter ( bag1 promoter -mNG) , selected under pyrimethamine (pyr) for 3 cycles in HFF cells in standard conditions. Cultures were exposed to alkaline stress to induce differentiation for 48hrs and parasites were then FACS sorted for mNG expression and prepared for sequencing. For in vivo screen; the same sub-library was transfected into Pru:Cas9 and selected over 3 cycles on pyr for stable integration of plasmids and then injected into cohorts of Swiss mice. At day 4 post infection peritoneal exudate containing Toxoplasma was collected. At day 35 brains of mice were collected. At each step DNA was extracted and guide <t>sequences</t> <t>amplified</t> for sequencing. (B) In vitro CRISPR screen analysis; Guide frequencies at the gene level, comparing FACS sorted RFP + mNG + (Sample 3)(y-axis) and RFP + mNG - (Sample 4)(x-axis) back to the initial population (Sample 2). Each data point represents a single gene and are coloured based on predicted (or known) function, with corresponding key. (C) Generation of individual Toxoplasma mutant strains; Guide plasmids were randomly picked from sub-library and used to generate individual mutant transgenic strains in the ME49:Cas9 reporter strain . Each mutant was subjected to alkaline stress for 48hrs and FACS used to analyze differentiation into bag1 promoter -mNG expressing parasites. Samples were then normalized to parental strain. BFD1 and BFD2 were used as controls. (D) Correlation of CRISPR screen results with differentiation of individual mutants. R 2 value calculated by linear regression. (E) Results of the mouse in vivo CRISPR screen, comparing Peritoneum Fitness Score (Log 2 FC (Sample 5 vs Sample 2) (x) vs Latency Fitness Score (Log 2 FC(Sample 6 vs 5))(y) Each data point represents a single gene, colour-coded as in B. (F) Comparison of in vitro and in vivo CRISPR screens, comparing the latency fitness score (as outlined in E) vs Log 2 FC(Sample 3 vs 2) (as in B). Each data point represents a single gene colour-coded as in B.
Crispr Library, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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(A) Schematic representation of sub-library CRISPR screens both in vitro and in vivo (mouse). Guide libraries targeting 66 genes (including controls) of 5 guides per gene, were constructed. For in vitro screen; the guide library was transfected into ME49:Cas9 reporter line, which expresses RFP constitutively using the Gra1 promoter ( gra1 promoter -RFP)and mNeonGreen (mNG) under control of the bradyzoite-specific BAG1 promoter ( bag1 promoter -mNG) , selected under pyrimethamine (pyr) for 3 cycles in HFF cells in standard conditions. Cultures were exposed to alkaline stress to induce differentiation for 48hrs and parasites were then FACS sorted for mNG expression and prepared for sequencing. For in vivo screen; the same sub-library was transfected into Pru:Cas9 and selected over 3 cycles on pyr for stable integration of plasmids and then injected into cohorts of Swiss mice. At day 4 post infection peritoneal exudate containing Toxoplasma was collected. At day 35 brains of mice were collected. At each step DNA was extracted and guide sequences amplified for sequencing. (B) In vitro CRISPR screen analysis; Guide frequencies at the gene level, comparing FACS sorted RFP + mNG + (Sample 3)(y-axis) and RFP + mNG - (Sample 4)(x-axis) back to the initial population (Sample 2). Each data point represents a single gene and are coloured based on predicted (or known) function, with corresponding key. (C) Generation of individual Toxoplasma mutant strains; Guide plasmids were randomly picked from sub-library and used to generate individual mutant transgenic strains in the ME49:Cas9 reporter strain . Each mutant was subjected to alkaline stress for 48hrs and FACS used to analyze differentiation into bag1 promoter -mNG expressing parasites. Samples were then normalized to parental strain. BFD1 and BFD2 were used as controls. (D) Correlation of CRISPR screen results with differentiation of individual mutants. R 2 value calculated by linear regression. (E) Results of the mouse in vivo CRISPR screen, comparing Peritoneum Fitness Score (Log 2 FC (Sample 5 vs Sample 2) (x) vs Latency Fitness Score (Log 2 FC(Sample 6 vs 5))(y) Each data point represents a single gene, colour-coded as in B. (F) Comparison of in vitro and in vivo CRISPR screens, comparing the latency fitness score (as outlined in E) vs Log 2 FC(Sample 3 vs 2) (as in B). Each data point represents a single gene colour-coded as in B.

Journal: bioRxiv

Article Title: Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

doi: 10.1101/2025.07.27.667068

Figure Lengend Snippet: (A) Schematic representation of sub-library CRISPR screens both in vitro and in vivo (mouse). Guide libraries targeting 66 genes (including controls) of 5 guides per gene, were constructed. For in vitro screen; the guide library was transfected into ME49:Cas9 reporter line, which expresses RFP constitutively using the Gra1 promoter ( gra1 promoter -RFP)and mNeonGreen (mNG) under control of the bradyzoite-specific BAG1 promoter ( bag1 promoter -mNG) , selected under pyrimethamine (pyr) for 3 cycles in HFF cells in standard conditions. Cultures were exposed to alkaline stress to induce differentiation for 48hrs and parasites were then FACS sorted for mNG expression and prepared for sequencing. For in vivo screen; the same sub-library was transfected into Pru:Cas9 and selected over 3 cycles on pyr for stable integration of plasmids and then injected into cohorts of Swiss mice. At day 4 post infection peritoneal exudate containing Toxoplasma was collected. At day 35 brains of mice were collected. At each step DNA was extracted and guide sequences amplified for sequencing. (B) In vitro CRISPR screen analysis; Guide frequencies at the gene level, comparing FACS sorted RFP + mNG + (Sample 3)(y-axis) and RFP + mNG - (Sample 4)(x-axis) back to the initial population (Sample 2). Each data point represents a single gene and are coloured based on predicted (or known) function, with corresponding key. (C) Generation of individual Toxoplasma mutant strains; Guide plasmids were randomly picked from sub-library and used to generate individual mutant transgenic strains in the ME49:Cas9 reporter strain . Each mutant was subjected to alkaline stress for 48hrs and FACS used to analyze differentiation into bag1 promoter -mNG expressing parasites. Samples were then normalized to parental strain. BFD1 and BFD2 were used as controls. (D) Correlation of CRISPR screen results with differentiation of individual mutants. R 2 value calculated by linear regression. (E) Results of the mouse in vivo CRISPR screen, comparing Peritoneum Fitness Score (Log 2 FC (Sample 5 vs Sample 2) (x) vs Latency Fitness Score (Log 2 FC(Sample 6 vs 5))(y) Each data point represents a single gene, colour-coded as in B. (F) Comparison of in vitro and in vivo CRISPR screens, comparing the latency fitness score (as outlined in E) vs Log 2 FC(Sample 3 vs 2) (as in B). Each data point represents a single gene colour-coded as in B.

Article Snippet: PCR reactions were then performed to amplify the guide region from the genomic DNAs as well as from the original CRISPR library (Addgene #80636) and amplified library used for transfections.

Techniques: CRISPR, In Vitro, In Vivo, Construct, Transfection, Control, Expressing, Sequencing, Injection, Infection, Amplification, Mutagenesis, Transgenic Assay, Comparison

(A) CRISPR screen results comparing back to input library (Sample 1), showing highly fitness conferring genes that were added to the library (as determined by ). Each datapoint is an individual gene and colour coded according to the key. (B) Surveyor assay to assess gene disruption across mutants generate in as compared to parental line. (C) Waterfall plot of Peritoneum Fitness Scores (Log 2 FC(Sample 5 vs 2). n=3 biological repeats, mean+/− SD shown. (D) Waterfall plot of Latency Fitness Scores (Log 2 FC(Sample 6 vs 5). n=3 biological repeats, mean+/− SD shown.

Journal: bioRxiv

Article Title: Differentiation of Toxoplasma into latent forms is linked to central carbon metabolism and requires a GID/CTLH-type E3 ubiquitin ligase

doi: 10.1101/2025.07.27.667068

Figure Lengend Snippet: (A) CRISPR screen results comparing back to input library (Sample 1), showing highly fitness conferring genes that were added to the library (as determined by ). Each datapoint is an individual gene and colour coded according to the key. (B) Surveyor assay to assess gene disruption across mutants generate in as compared to parental line. (C) Waterfall plot of Peritoneum Fitness Scores (Log 2 FC(Sample 5 vs 2). n=3 biological repeats, mean+/− SD shown. (D) Waterfall plot of Latency Fitness Scores (Log 2 FC(Sample 6 vs 5). n=3 biological repeats, mean+/− SD shown.

Article Snippet: PCR reactions were then performed to amplify the guide region from the genomic DNAs as well as from the original CRISPR library (Addgene #80636) and amplified library used for transfections.

Techniques: CRISPR, Disruption