Protocol for genome-scale CRISPR screening in engineered lineage reporter hPSCs to study cell fate determination

Abstract

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).

Keywords: CRISPR; Cell Differentiation; High Throughput Screening; Stem Cells.

Graphical abstract

Figure 1

Construction of inducible Cas9 expression systems in PAX6-tdTomato reporter hPSCs (A) Schematic view of the targeting strategy of PAX6-tdTomato reporter cell line through gRNA-guided CRISPR/Cas9 system. ATG, start codon; E, exon; PA, poly (A) signal; Neo, neomycin resistance gene. (B) Schematic diagram for constructing inducible Cas9 (iCas9) expression cassette in PAX6-tdTomato line through TALEN (marked in blue bars)-mediated gene targeting at the AAVS1 loci. CAG, CMV enhancer/chicken β-actin promoter; M2rtTA, reverse tetracycline transactivator; TRE, tetracycline response element, PA, poly (A) signal. (C) Immunostaining results show that Dox treatment induces Cas9 expression, but not affects the pluripotent marker (NANOG) expression in PAX6-tdTomato/iCas9 hPSCs. Antibody dilutions for FLAG and NANOG are 1:1,000 and 1:500, respectively. Scale bar, 75 μm.

Figure 2

Titration of packaged lentivirus sgRNA library (A) Titration the optimal concentration of puromycin for hPSCs. 0, 0.25, 0.34, 0.5, 1 and 5 μg/mL puromycin were applied in hPSCs for 3 days, and the killing efficiency was observed under the microscope. Scale bar, 100 μm. (B) Phase contrast images show that HEK293FT cells in the control well without puromycin treatment reach 70% confluency 4 days after passaging. In puromycin treated wells, all cells in the well infected with 0 μL virus solution are killed, and the survival rates of cells infected with 2.5, 5, 10 and 20 μL virus solution show gradient increase. Scale bar, 100 μm.

Figure 3

Genome-scale CRISPR screening in PAX6-tdTomato/iCas9 hPSCs (A) Timeline of the screening procedure. 3.4 ×10⁶ hPSCs were infected with sgRNA lentiviral libraries of A pool or B pool and seeded in one MEF feeder-coated 6-well plate. 0.5 μg/mL puromycin was added from day 2 to day 5 to remove uninfected cells. MEF conditioned medium was supplied from day 3 to day 7. When reached 70% confluency at day 7, the library-infected hPSCs were passaged onto six 6-well plates with feeder layer. 2 days after passage, the hPSCs were treated with or without Dox (3 plates for each group) for 3 days. 5 days after passage, when the hPSCs were 70% confluent, both the control and Dox-treated cells were passaged onto eighteen 6-well plates with feeder layer. tdTomato positive cells organized into rosette-like structures were observable 6 days after the second passage, and FACS sorting and DNA extraction were performed. The whole procedure takes around 18 days. (B) Some hPSC colones express clustered tdTomato after Dox treatment. Scale bar, 100 μm. (C) Fluorescent images show that FACS-sorted cells have uniform tdTomato expression. Scale bar, 100 μm.