Develop a pooled CRISPR-Cas9 screening strategy using primary human hematopoietic stem/progenitor cells (HSPCs) to identify genes that regulate erythroblast enucleation.
Approach:
Cell model: Human CD34+ bone marrow HSPCs were differentiated ex vivo into erythroblasts and cultured with stromal cells.
Screen: Cells were transduced with a pooled sgRNA library, edited with Cas9, and enucleated cells were isolated by flow sorting. sgRNA abundance was measured by sequencing RNA-derived cDNA.
Analysis: Guide and gene enrichment were assessed by comparing unsorted and sorted populations, with false discovery rates calculated using the Benjamini-Hochberg procedure.
Interpretation:
The provided context describes the study rationale and methods but does not report screening results.
Limitations:
The supplied context ends during the methods section and contains no results or study limitations.
