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

摘要

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).

材料

货号

品牌

产品描述

F3165

Sigma-Aldrich

单克隆抗-FLAG^® M2 小鼠抗, 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)

HVHP02500

Millipore

Durapore^® 膜过滤装置, 0.45 µm, Durapore^®, filter diam. 25 mm, hydrophobic

540411

Sigma-Aldrich

嘌呤霉素，二盐酸盐，经细胞培养测试, Puromycin, CAS 58-58-2, is a protein synthesis inhibitor that causes premature release of nascent polypeptide chains.