Single-cell deletion analyses show control of pro–T cell developmental speed and pathways by Tcf7, Spi1, Gata3, Bcl11a, Erg, and Bcl11b.

As early T cell precursors transition from multipotentiality to T lineage commitment, they change expression of multiple transcription factors. It is unclear whether individual transcription factors directly control choices between T cell identity and some alternative fate or whether these factors mostly affect proliferation or survival during the normal commitment process. Here, we unraveled the impacts of deleting individual transcription factors at two stages in early T cell development, using synchronized in vitro differentiation systems, single-cell RNA-seq with batch indexing, and controlled gene-disruption strategies. First, using a customized method for single-cell CRISPR disruption, we defined how the early-acting transcription factors Bcl11a, Erg, Spi1 (PU.1), Gata3, and Tcf7 (TCF1) function before commitment. The results revealed a kinetic tug of war within individual cells between T cell factors Tcf7 and Gata3 and progenitor factors Spi1 and Bcl11a, with an unexpected guidance role for Erg. Second, we tested how activation of transcription factor Bcl11b during commitment altered ongoing cellular programs. In knockout cells where Bcl11b expression was prevented, the cells did not undergo developmental arrest, instead following an alternative path as T lineage commitment was blocked. A stepwise, time-dependent regulatory cascade began with immediate-early transcription factor activation and E protein inhibition, finally leading Bcl11b knockout cells toward exit from the T cell pathway. Last, gene regulatory networks of transcription factor cross-regulation were extracted from the single-cell transcriptome results, characterizing the specification network operating before T lineage commitment and revealing its links to both the Bcl11b knockout alternative network and the network consolidating T cell identity during commitment. Knockout commitment: Many transcription factors are involved in the lineage commitment of early T cell precursors, yet how individual transcription factors alter early T cell development is not well defined. Here, Zhou et al. used CRISPR to knockout various transcription factors (Bcl11a, Erg, Spi1, Gata3, Tcf7, and Bcl11b) to determine their individual roles in T cell lineage progression via single-cell transcriptomics. They found that knockout of each of these genes altered the progression of T cells in similar and unique ways, allowing them to identify various gene regulatory networks linked to specific transcription factors. Specifically, they found that Bcl11b was involved in progenitors exiting the T cell pathway. Thus, this work provides a resource and method for studying the dynamics of various transcription factors during T cell lineage commitment. [ABSTRACT FROM AUTHOR]