- Protocol for genome-scale CRISPR screening in engineered lineage reporter hPSCs to study cell fate determination.
Protocol for genome-scale CRISPR screening in engineered lineage reporter hPSCs to study cell fate determination.
STAR protocols (2021-06-08)
Zhongshu Zhou, Lin Ma, Xiaoqing Zhang
PMID34095862
RESUMO
PAX6 is a key determinant of human neuroectoderm cell fate. Here, we describe a protocol for genome-scale CRISPR screening for use in genetically engineered human pluripotent stem cells (hPSCs). Using the germ layer reporter PAX6 and an inducible CRISPR/Cas9 knockout system, we describe how to identify lineage-specific preventing genes. This protocol can be applied for use with other reporter genes to study cell fate determination in hPSCs. For complete details on the use and execution of this protocol, please refer to Xu et al. (2021).
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Sigma-Aldrich
ANTI-FLAG® M2 monoclonal, clone M2, purified immunoglobulin (Purified IgG1 subclass), buffered aqueous solution (10 mM sodium phosphate, 150 mM NaCl, pH 7.4, containing 0.02% sodium azide)
Millipore
Filtro de membrana de PVDF, tamanho de poro de 0,45 μm, Durapore®, filter diam. 25 mm, hydrophobic