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标题:Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening
时间:2021-04-02 09:12:26
DOI:10.1038/nprot.2017.016
PMID:28333914
大小:1092 kb
页数:73 PAGES
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目录:
  • Abstract
  • INTRODUCTION
    • Cas9 as a tool for precise genome editing
    • Transcriptional activation with Cas9
    • Applications of CRISPR-Cas9 screening
    • Comparison with other pooled screening technologies
    • Experimental Design
      • Screening strategies
        • Design and selection of the sgRNA library
        • Approaches for sgRNA library construction and delivery
        • Selection
        • Analysis of screening results
        • Validation of candidate genes
  • MATERIALS
    • REAGENTS
      • SgRNA libraries and backbones
      • Custom sgRNA library cloning
      • SgRNA plasmid amplification
      • Next-generation sequencing
      • Mammalian cell culture
      • Lentivirus production and titer
      • Screening and Validation
    • EQUIPMENT
    • REAGENT SETUP
      • TBE electrophoresis solution
      • Ethanol, 80% (vol/vol)
      • D10 medium
      • mTeSR1 medium
      • Proteinase K, 300 U ml−1
      • Deoxyribonuclease I, 50 KU ml−1
      • RNA lysis buffer
      • EGTA, 0.5 M, pH 8.3
      • RNA lysis stop solution
      • Oligo dT, 100 μM
    • EQUIPMENT SETUP
      • Large LB agar plates (245 mm square bioassay dish, ampicillin)
      • Standard LB agar plates (100 mm Petri dish, ampicillin)
    • PROCEDURE
      • Designing a custom sgRNA library o TIMING 3–5 w; 1 w hands-on
      • Cloning a custom sgRNA library o TIMING 2 d
      • Amplification of pooled sgRNA library o TIMING 2 d
      • Next-generation sequencing of the amplified sgRNA library to determine sgRNA distribution o TIMING 3–5 d
      • Lentivirus production and titer o TIMING 1–2 w
      • Lentiviral transduction and screening o TIMING 3–6 w
      • Harvest genomic DNA for screening analysis o TIMING 5–7 d
      • Validation of candidate genes for screening phenotype o TIMING 4–5 w
      • Troubleshooting
      • Timing
  • ANTICIPATED RESULTS
  • References
  • Figure 1
  • Figure 2
  • Figure 3
  • Figure 4
  • Table 1
  • Table 2
  • Table 3
  • Table 4
  • Table 5
  • Table 6

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