Base-editing screens illuminate variant effects in human hematopoiesis

Abstract

In a recent issue of Cell, Martin-Rufino et al. develop a strategy for performing high-throughput base-editing CRISPR screens coupled with single-cell readouts in the context of human hematopoiesis. Through a series of proof-of-principle experiments, the authors demonstrate the potential of base-editing screens for the study and treatment of hematological disorders.

Figure 1

High-throughput base-editing screens in human hematopoiesis Schematic of the protocol employed for variant screening in primary hematopoietic stem and progenitor cells (HSPCs) and different hematopoietic lineages (top). HSPCs isolated from healthy donors are transduced with a lentiviral sgRNA library targeting the gene(s) of interest, followed by electroporation of base editors. Then, HSPC maintenance media can be substituted with differentiation media to promote hematopoiesis. Hematopoietic stem cells (HSCs) represent one of the subgroups within HSPCs that possess self-renewal and multilineage differentiation capacity. MPP (multipotent progenitor), LMPP (lymphoid-primed multipotential progenitor), CLP (common lymphoid progenitor), CMP (common myeloid progenitor), GMP (granulocyte–monocyte progenitors), and MEP (megakaryocyte–erythrocyte progenitors) are also shown. The effect of nucleotide variants in hematopoiesis is determined using (1) functional assays, such as FACS; (2) single-cell RNA sequencing (scRNA-seq); and (3) single-cell genotyping (bottom left). Findings of base-editing screens in hematopoiesis (1) can advance cell therapies by improving cell engineering, (2) allow the modulation of gene expression through the editing of gene regulatory elements, and (3) facilitate the interpretation of the effects of nucleotide variants on gene function, including the classification of VUSs (bottom right). Figure created using BioRender (https://biorender.com).