CRISPR Efficiency Evaluation in Reporter Cells

TLR reporter cell line were pre-seeded and transfected with various Cas9 constructs and sgRNA with or without GFP donor template (Addgene plasmid #31475¹⁹ (link)). Flow cytometry analysis of GFP-positive cells was performed using CytoFLEX cell analyzer (Beckman Coulter) to assess HDR efficiency. At least 50,000 cells from each well were analyzed. To assess NHEJ efficiency, mCherry signal was analyzed using Harmony 3.5 (PerkinElmer) after image acquisition with Operetta High Content Screening system (PerkinElmer).

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Zhao C., Zhao Y., Zhang J., Lu J., Chen L., Zhang Y., Ying Y., Xu J., Wei S, & Wang Y. (2018). HIT-Cas9: A CRISPR/Cas9 Genome-Editing Device under Tight and Effective Drug Control. Molecular Therapy. Nucleic Acids, 13, 208-219.

Publication 2018

CytoFLEX cell analyzer Harmony 3.5 Operetta High Content Screening system

After image Cell line Cells Donor Flow cytometry Nhej Plasmid

Corresponding Organization : University of Chinese Academy of Sciences

Other organizations : China Agricultural University

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Variable analysis

independent variables

Cas9 constructs
SgRNA
GFP donor template (Addgene plasmid #31475)

dependent variables

Percentage of GFP-positive cells (HDR efficiency)
MCherry signal (NHEJ efficiency)

control variables

TLR reporter cell line
Number of cells analyzed (at least 50,000 cells per well)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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