SUMMARY Our understanding of the mechanisms that regulate hematopoietic stem/progenitor cells (HSPCs) has been advanced by the ability to genetically manipulate mice; however, germline modification is time-consuming and expensive. Here we describe fast, efficient, and cost-effective methods to directly modify the genomes of mouse and human HSPCs using the CRISPR/Cas9 system. Using plasmid and virus-free delivery of guide RNAs alone into Cas9-expressing HSPCs, or Cas9-guide-RNA ribonucleoprotein (RNP) complexes into wild-type cells, we have achieved extremely efficient gene disruption in primary HSPCs from mouse (>60%) and human (~75%). These techniques enabled rapid evaluation of the functional effects of gene loss of Eed, Suz12, and DNMT3A. We also achieved homology-directed repair in primary human HSPCs (>20%). These methods will significantly expand applications for CRISPR/Cas9 technologies for studying normal and malignant hematopoiesis.

SUMMARY How cancer cells adapt to metabolically adverse conditions in patients and strive to proliferate is a fundamental question in cancer biology. Here we show that AMP-activated protein kinase (AMPK), a metabolic checkpoint kinase, confers metabolic stress resistance to leukemia-initiating cells (LICs) and promotes leukemogenesis. Upon dietary restriction, MLL-AF9-induced murine AML activated AMPK and maintained leukemogenic potential. AMPK deletion significantly delayed leukemogenesis and depleted LICs by reducing the expression of glucose transporter 1 (Glut1), compromising glucose flux, and increasing oxidative stress and DNA damage. LICs were particularly dependent on AMPK to suppress oxidative stress in the hypoglycemic bone marrow environment. Strikingly, AMPK inhibition synergized with physiological metabolic stress caused by dietary restriction and profoundly suppressed leukemogenesis. Our results indicate that AMPK protects LICs from metabolic stress, and that combining AMPK inhibition with physiological metabolic stress potently suppresses AML by inducing oxidative stress and DNA damage.

Adoptive cell therapy with viral-specific T cells has been successfully used to treat life-threatening viral infections, supporting the application of this approach against COVID-19. We expand SARS-CoV-2 T-cells from the peripheral blood of COVID-19-recovered donors and non-exposed controls using different culture conditions. We observe that the choice of cytokines modulates the expansion, phenotype and hierarchy of antigenic recognition by SARS-CoV-2 T-cells. Culture with IL-2/4/7 but not other cytokine-driven conditions result in >1000 fold expansion in SARS-CoV-2 T-cells with a retained phenotype, function and hierarchy of antigenic recognition when compared to baseline (pre-expansion) samples. Expanded CTLs are directed against structural SARS-CoV-2 proteins, including the receptor-binding domain of Spike. SARS-CoV-2 T-cells cannot be efficiently expanded from the peripheral blood of non-exposed controls. Since corticosteroids are used for the management of severe COVID-19, we propose an efficient strategy to inactivate the glucocorticoid receptor gene ( NR3C1 ) in SARS-CoV-2 CTLs using CRISPR-Cas9 gene editing.

The piggyBac transposon is one of the most attractive nonviral tools for mammalian genome manipulations. Given that piggybac mobilizes in a "cut-and-paste" fashion, integrant remobilization could potentially damage the host genome. Here, we report a novel piggyBac transposon system with a series of recombinant transposases. We found that the transposition activity of wild-type (PBase) and hyperactive (hyPBase) piggyBac transposases can be significantly increased by peptide fusions in a cell-type dependent fashion, with the greatest change typically seen in mouse embryonic stem (ES) cells. The two most potent recombinant transposases, TPLGMH and ThyPLGMH, give a 9- and 7-fold increase, respectively, in the number of integrants in HEK293 compared with Myc-tagged PBase (MycPBase), and both display 4-fold increase in generating induced pluripotential stem cells. Interestingly, ThyPLGMH but not TPLGMH shows improved chromosomal excision activity (2.5-fold). This unique feature of TPLGMH provides the first evidence that integration activity of a transposase can be drastically improved without increasing its remobilization activity. Transposition catalyzed by ThyPLGMH is more random and occurs further from CpG islands than that catalyzed by MycPBase or TPLGMH. Our transposon system diversifies the mammalian genetic toolbox and provides a spectrum of piggyBac transposases that is better suited to different experimental purposes.

Histone variants contribute to the complexity of the chromatin landscape and play an integral role in defining DNA domains and regulating gene expression. The histone H3 variant H3.3 is incorporated into genic elements independent of DNA replication by its chaperone HIRA. Here we demonstrate that Hira is required for the self-renewal of adult hematopoietic stem cells (HSCs) and to restrain erythroid differentiation. Deletion of Hira led to rapid depletion of HSCs while differentiated hematopoietic cells remained largely unaffected. Depletion of HSCs after Hira deletion was accompanied by increased expression of bivalent and erythroid genes, which was exacerbated upon cell division and paralleled increased erythroid differentiation. Assessing H3.3 occupancy identified a subset of polycomb-repressed chromatin in HSCs that depends on HIRA to maintain the inaccessible, H3.3-occupied state for gene repression. HIRA-dependent H3.3 incorporation thus defines distinct repressive chromatin that represses erythroid differentiation of HSCs.