Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification.

SUMMARY Vitamin D insufficiency is a worldwide epidemic affecting billions of individuals, including pregnant women and children. Despite its high incidence, the impact of active vitamin D3 (1,25(OH)D3) on embryonic development beyond osteo-regulation remains largely undefined. Here, we demonstrate that 1,25(OH)D3 availability modulates zebrafish hematopoietic stem and progenitor cell (HSPC) production. Loss of Cyp27b1-mediated biosynthesis or vitamin D receptor (VDR) function by gene knockdown resulted in significantly reduced runx1 expression and Flk1+cMyb+ HSPC numbers. Selective modulation in vivo and in vitro in zebrafish indicated that vitamin D3 acts directly on HSPCs, independent of calcium regulation, to increase proliferation. Notably, ex vivo treatment of human HSPCs with 1,25(OH)D3 also enhanced hematopoietic colony numbers, illustrating conservation across species. Finally, gene expression and epistasis analysis indicated that CXCL8 (IL-8) was a functional target of vitamin D3-mediated HSPC regulation. Together, these findings highlight the relevance of developmental 1,25(OH)D3 availability for definitive hematopoiesis and suggest potential therapeutic utility in HSPC expansion.

A single-agent dose-response database can be derived from historic literature that enables comparison of the dose-intensity of all combination regimens on one scale. The dose-intensity increase required to improve outcome can then be identified in earlier trials that tested that variable. SDI methodology should be tested prospectively in contemporary patients, and may be useful in guiding dosage increases beyond the conventional range.

A B S T R A C T Pulmonary hemodynamics and Rotta, Canepa, Hurtado, Velasquez, and Chavez in 1956 (1) and has been confirmed many times since, both in acclimatized natives (2, 3) and in newcomers (4). The relationship of this pulmonary hypertension to the development of high altitude pulmonary edema (HAPE) is uncertain, but pulmonary artery pressure is higher during episodes of HAPE than during control observations in the same individuals (5), and persons who have had such episodes previously have higher pulmonary artery pressures when reexposed to altitude than nonsusceptible individuals under the same conditions (6). Although it is recognized that the peak incidence of HAPE occurs 1-3 days after entry to high altitude (7), it has not been established whether pulmonary arterial pressure rises in a progressive fashion over the same time course. A progressive rise in pulmonary arterial pressure during a 6 wk period at 12,700 ft has been reported in steers, apparently associated with the right heart failure termed "brisket disease" (8), but was not observed in lambs in a matched experiment (9). Therefore, we decided to investigate in ourselves the time course of the pulmonary arterial pressure both at rest and during exercise at 12,470 ft and to examine other aspects of cardiovascular function over the same time span. We also explored the possibility suggested by Haab, Held, Ernst, and Farhi (10) and Reeves, Halpin, Cohn, and Daoud (11) that hypoxia widens the alveolar to arterial 02 difference.

The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro–B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro– to pre–B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1’s critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.