knockout toxoplasma Search Results


lourido toxoplasma crispr library v1  (Addgene inc)
92
Addgene inc lourido toxoplasma crispr library v1
Figure 1 | Schematic of the <t>CRISPR</t> screening procedure. (a) sgRNA libraries are generated by massively parallel oligonucleotide synthesis and cloned into the screening vector while maintaining sequence diversity (Supplementary Methods). Before each screen, the RH/Cas9 strain is checked to ensure Cas9 expression (Supplementary Methods) and high gene-disruption efficiency (Steps 1–25). The CRISPR library is linearized to maximize integration into the parasite genome (Steps 26–28). In preparation for the screen, HFF cells and parasites are expanded to achieve sufficient library coverage (Steps 29 and 30). The linearized library is introduced into the RH/Cas9 parasites by electroporation (Steps 31–37), and the population is grown over three lytic cycles in HFF cells (Steps 38–51), at which point further cycles of selection are optional (Steps 52–55). Genomic DNA is extracted from the resulting populations (Step 56), and sgRNAs are amplified from this genomic DNA and from the input library by PCR (Steps 57–59). The Illumina sequencing adaptors P5 and P7, and multiplexing indices are added during PCR. The abundances of sgRNAs in the transfected library relative to all subsequent samples are quantified using next- generation sequencing and custom analysis pipelines (Steps 60–66). Genes that confer fitness will have a lower abundance in the populations after growth or selection relative to the transfected CRISPR library. (b) Diagram of the amplification of sgRNAs from the CRISPR library before sequencing. The sequence of the amplicon is illustrated with Ns denoting the barcode for multiplexing (gray) and the sgRNA (blue). Regions or homology to the vector (orange and green), and Illumina sequencing adaptors (red) are also highlighted.
Lourido Toxoplasma Crispr Library V1, 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
https://www.bioz.com/result/lourido toxoplasma crispr library v1/product/Addgene inc
Average 92 stars, based on 1 article reviews
lourido toxoplasma crispr library v1 - by Bioz Stars, 2026-04
92/100 stars
  Buy from Supplier

Image Search Results


Figure 1 | Schematic of the CRISPR screening procedure. (a) sgRNA libraries are generated by massively parallel oligonucleotide synthesis and cloned into the screening vector while maintaining sequence diversity (Supplementary Methods). Before each screen, the RH/Cas9 strain is checked to ensure Cas9 expression (Supplementary Methods) and high gene-disruption efficiency (Steps 1–25). The CRISPR library is linearized to maximize integration into the parasite genome (Steps 26–28). In preparation for the screen, HFF cells and parasites are expanded to achieve sufficient library coverage (Steps 29 and 30). The linearized library is introduced into the RH/Cas9 parasites by electroporation (Steps 31–37), and the population is grown over three lytic cycles in HFF cells (Steps 38–51), at which point further cycles of selection are optional (Steps 52–55). Genomic DNA is extracted from the resulting populations (Step 56), and sgRNAs are amplified from this genomic DNA and from the input library by PCR (Steps 57–59). The Illumina sequencing adaptors P5 and P7, and multiplexing indices are added during PCR. The abundances of sgRNAs in the transfected library relative to all subsequent samples are quantified using next- generation sequencing and custom analysis pipelines (Steps 60–66). Genes that confer fitness will have a lower abundance in the populations after growth or selection relative to the transfected CRISPR library. (b) Diagram of the amplification of sgRNAs from the CRISPR library before sequencing. The sequence of the amplicon is illustrated with Ns denoting the barcode for multiplexing (gray) and the sgRNA (blue). Regions or homology to the vector (orange and green), and Illumina sequencing adaptors (red) are also highlighted.

Journal: Nature Protocols

Article Title: CRISPR-Cas9-based genome-wide screening of Toxoplasma gondii

doi: 10.1038/nprot.2017.131

Figure Lengend Snippet: Figure 1 | Schematic of the CRISPR screening procedure. (a) sgRNA libraries are generated by massively parallel oligonucleotide synthesis and cloned into the screening vector while maintaining sequence diversity (Supplementary Methods). Before each screen, the RH/Cas9 strain is checked to ensure Cas9 expression (Supplementary Methods) and high gene-disruption efficiency (Steps 1–25). The CRISPR library is linearized to maximize integration into the parasite genome (Steps 26–28). In preparation for the screen, HFF cells and parasites are expanded to achieve sufficient library coverage (Steps 29 and 30). The linearized library is introduced into the RH/Cas9 parasites by electroporation (Steps 31–37), and the population is grown over three lytic cycles in HFF cells (Steps 38–51), at which point further cycles of selection are optional (Steps 52–55). Genomic DNA is extracted from the resulting populations (Step 56), and sgRNAs are amplified from this genomic DNA and from the input library by PCR (Steps 57–59). The Illumina sequencing adaptors P5 and P7, and multiplexing indices are added during PCR. The abundances of sgRNAs in the transfected library relative to all subsequent samples are quantified using next- generation sequencing and custom analysis pipelines (Steps 60–66). Genes that confer fitness will have a lower abundance in the populations after growth or selection relative to the transfected CRISPR library. (b) Diagram of the amplification of sgRNAs from the CRISPR library before sequencing. The sequence of the amplicon is illustrated with Ns denoting the barcode for multiplexing (gray) and the sgRNA (blue). Regions or homology to the vector (orange and green), and Illumina sequencing adaptors (red) are also highlighted.

Article Snippet: pU6-SAG1 (Addgene, cat. no. 80322) Lourido Toxoplasma CRISPR Library V1 (Addgene, cat. no. 80636) pU6-DHFR (Addgene, cat. no. 80329) pCas9-CAT (Addgene, cat. no. 80323) pU6-Decoy (Addgene, cat. no. 80324) Ampicillin (Sigma-Aldrich, cat. no. A0166-25G) Glycerol (EMD, cat. no. GX0185-6) Prolong Gold (Invitrogen, cat. no. P1044) Molecular biology-grade water (e.g., from an EMD Millipore Milli-Q Integral System; Fisher Scientific, cat. no. ZRXQ003US) Gentamicin (Gibco, cat. no. 15710-064) EGTA (Sigma-Aldrich, cat. no. E4378-500G) HEPES (Sigma-Aldrich, cat. no. 3375-1KG) KH2PO4 (Sigma-Aldrich, cat. no. P5655) K2HPO4 (Sigma-Aldrich, cat. no. 1551128) KCl (Fisher Scientific, cat. no. SP138-500) MgCl2 (Mallinckrodt, cat. no. 5958) Methanol for HPLC gradient analysis (Millipore, cat. no. 67-56-1) EDTA (Amresco, cat. no. 0322-500G) Tris base (Sigma-Aldrich, cat. no. TRIS-RO) • • • • • • • • • • • • • • • • • • • Glacial acetic acid (Spectrum, cat. no. A1010) ! cautIon Glacial acetic acid is corrosive; avoid contact with eyes, skin, and clothing; wear protective gloves while handling.

Techniques: CRISPR, Generated, Oligonucleotide Synthesis, Clone Assay, Plasmid Preparation, Sequencing, Expressing, Disruption, Electroporation, Selection, Amplification, Illumina Sequencing, Multiplexing, Transfection, Next-Generation Sequencing