The CRISPR system is widely used in genome editing for biomedical research. Here, using either dual paired Cas9D10A nickases or paired Cas9 nuclease we characterize unintended larger deletions at on-target sites that frequently evade common genotyping practices. We found that unintended larger deletions are prevalent at multiple distinct loci on different chromosomes, in cultured cells and mouse embryos alike. We observed a high frequency of microhomologies at larger deletion breakpoint junctions, suggesting the involvement of microhomology-mediated end joining in their generation. In populations of edited cells, the distribution of larger deletion sizes is dependent on proximity to sgRNAs and cannot be predicted by microhomology sequences alone.

IntroductionThe junctional adhesion molecules (JAMs) are a subset of the immunoglobulin (Ig) protein superfamily and are characterized by the presence of both a type V and a type C2 extracellular Ig domain. JAMs and related molecules have been involved in the control of interendothelial junctions and leukocyte transendothelial migration through homotypic and heterotypic interactions. 1-5 JAM-B has been previously shown to interact with JAM-C and contributes to leukoendothelial and interendothelial cell-cell adhesion. 6,7 In mice, JAM-B expression is restricted to endothelial cells whereas JAM-C is expressed by various cell types including endothelial, 1,2 fibroblastic, 8 and smooth muscle cells. 9 Moreover, we have recently shown that JAM-C expression in lymph node fibroblastic cells is required for constitutive secretion of several chemokines such as SDF-1␣. 10 Recently, expression of several JAM family members, such as JAM-A, JAM-C, JAM4, or ESAM, has been reported in hematopoietic stem cells (HSCs), although a function for these proteins in hematopoiesis remains unknown. [11][12][13][14][15][16] In adult mammals, HSCs are rare cells mainly located in the bone marrow (BM) and able to generate all mature blood cells. In mice, HSCs are comprised within the LSK compartment as defined by the Lineage Neg c-kit Hi Sca-1 Hi (LSK) phenotype; the LSK compartment can be further subdivided using additional markers such as CD34, CD150, or CD48. Indeed, CD34 Neg CD135 Neg LSK, or Thy-1 Lo LSK 17,18 and CD150 Pos CD48 Neg LSK cells 19 have been shown to contain, respectively, around 20% and 50% of HSCs with long-term hematopoietic reconstitution potential (LT-HSCs). Because HSCs give rise to mature hematopoietic cells, including immune cells, their replacement must be adjusted to homeostatic or stress conditions such as infections, inflammation, or blood loss, and their expansion must be controlled to avoid exhaustion through inappropriate proliferation and differentiation. This is possible by the coordinated regulation of quiescence, self-renewal, and differentiation of HSCs through appropriate signals delivered by functional microenvironments called niches. [20][21][22] HSCs are restrained in these specialized microenvironments by interactions mediated by adhesion molecules and chemokine receptors expressed by HSCs, such as VLA-4 or CXCR4, with their ligands present within the BM microenvironment. Other signaling pathways controlled by growth factors and their receptors expressed on HSCs, such as angiopoietin-1 and TIE2, SCF and KIT, Thrombopoietin and MPL, have also been involved in HSC maintenance and retention in BM niches. 23,24 Whether the JAM family members expressed on HSCs also contribute to this signaling network has not been addressed so far.When necessary, HSCs enter the cell cycle to maintain BM cellularity and replenish peripheral blood in a process called mobilization, which can be induced using various hematopoietic growth factors, cyclophosphamide in combination with G-CSF (Cy/G-CSF), or intravenous inject...

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivo AGM and yolk sac endothelial niche.

The junctional adhesion molecules Jam‐b and Jam‐c interact together at interendothelial junctions and have been involved in the regulation of immune response, inflammation, and leukocyte migration. More recently, Jam‐c has been found to be expressed by hematopoietic stem and progenitor cells (HSPC) in mouse. Conversely, we have reported that Jam‐b is present on bone marrow stromal cells and that Jam‐b‐deficient mice have defects in the regulation of hematopoietic stem cell pool. In this study, we have addressed whether interaction between Jam‐b and Jam‐c participates to HSPC mobilization or hematopoietic reconstitution after irradiation. We show that a blocking monoclonal antibody directed against Jam‐c inhibits hematopoietic reconstitution, progenitor homing to the bone marrow, and induces HSPC mobilization in a Jam‐b dependent manner. In the latter setting, antibody treatment over a period of 3 days does not alter hematopoietic differentiation nor induce leukocytosis. Results are translated to human hematopoietic system in which a functional adhesive interaction between JAM‐B and JAM‐C is found between human HSPC and mesenchymal stem cells. Such an interaction does not occur between HSPC and human endothelial cells or osteoblasts. It is further shown that anti‐JAM‐C blocking antibody interferes with CD34+ hematopoietic progenitor homing in mouse bone marrow suggesting that monoclonal antibodies inhibiting JAM‐B/JAM‐C interaction may represent valuable therapeutic tools to improve stem cell mobilization protocols. Stem Cells 2014;32:1043–1054

Upon their interaction with cognate antigen, T cells integrate different extracellular and intracellular signals involving basal and induced protein–protein interactions, as well as the binding of proteins to lipids, which can lead to either cell activation or inhibition. Here, we show that the selective T cell expression of CMIP, a new adapter protein, by targeted transgenesis drives T cells toward a naïve phenotype. We found that CMIP inhibits activation of the Src kinases Fyn and Lck after CD3/CD28 costimulation and the subsequent localization of Fyn and Lck to LRs. Video microscopy analysis showed that CMIP blocks the recruitment of LAT and the lipid raft marker cholera toxin B at the site of TCR engagement. Proteomic analysis identified several protein clusters differentially modulated by CMIP and, notably, Cofilin-1, which is inactivated in CMIP-expressing T cells. Moreover, transgenic T cells exhibited the downregulation of GM3 synthase, a key enzyme involved in the biosynthesis of gangliosides. These results suggest that CMIP negatively impacts proximal signaling and cytoskeletal rearrangement and defines a new mechanism for the negative regulation of T cells that could be a therapeutic target.

Homeostasis is a word widely used in the scientific community to refer to the property of a system to maintain its uniformity and functionality. In living organisms, the word refers to the concept enunciated 150 years ago by C. Bernard by which external variations must be compensated for in order to maintain internal conditions compatible with life. This is especially true in the case of highly dynamic system such as the hematopoietic system that requires the coordinated control of cell proliferation and death within specialized microenvironments that are anatomically distinct. As a consequence, hematopoietic cell adhesion and migration must be tightly controlled in order for hematopoietic cells to reach and to be maintained in appropriate microenvironments. The junctional adhesion molecules (JAMs) are adhesion molecules that belong to the immunoglobulin superfamily (IgSf) and that have been initially identified as important players controlling vascular permeability and leukocyte transendothelial migration. This involves the regulated localization of the JAMs at lateral endothelial cell/cell borders and their interaction with leukocyte integrins. More recently, some of the JAM family members have also been found to be expressed by stromal cells and to regulate chemokine secretion within lymphoid organs, acting not only on leukocyte transendothelial migration, but also on hematopoietic cell retention within specialized microenvironments. This review summarizes recent progress in understanding the role of the JAMs in leukocyte adhesion and migration to tentatively draw an integrated view of the homeostatic function of the JAMs within the hematopoietic system.

a b s t r a c tMetastasis is a major clinical issue and results in poor prognosis for most cancers. The Junctional Adhesion Molecule-C (JAM-C) expressed by B16 melanoma and endothelial cells has been involved in metastasis of tumor cells through homophilic JAM-C/JAM-C trans-interactions. Here, we show that JAM-B expressed by endothelial cells contributes to murine B16 melanoma cells metastasis through its interaction with JAM-C on tumor cells. We further show that this adhesion molecular pair mediates melanoma cell adhesion to primary Lung Microvascular Endothelial Cells and that it is functional in vivo as demonstrated by the reduced metastasis of B16 cells in Jam-b deficient mice.

Summary Hematopoietic stem cells (HSCs) emerge during development from the vascular wall of the main embryonic arteries. The onset of circulation triggers several processes that provide critical external factors for HSC generation. Nevertheless, it is not fully understood how and when the onset of circulation affects HSC emergence. Here we show that in Ncx1 −/− mouse embryos devoid of circulation the HSC lineage develops until the phenotypic pro-HSC stage. However, these cells reside in an abnormal microenvironment, fail to activate the hematopoietic program downstream of Runx1, and are functionally impaired. Single-cell transcriptomics shows that during the endothelial-to-hematopoietic transition, Ncx1 −/− cells fail to undergo a glycolysis to oxidative phosphorylation metabolic switch present in wild-type cells. Interestingly, experimental activation of glycolysis results in decreased intraembryonic hematopoiesis. Our results suggest that the onset of circulation triggers metabolic changes that allow HSC generation to proceed.