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The efficiency of precise CRISPR/Cas9 genome editing is increased by inhibition of the nonhomologous end joining pathway. The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4–5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.

Background The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic ‘safe harbor’ site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8–11 kb inserts into Rosa26 of C57BL/6 zygotes. Results We found that 10–20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9. Conclusions Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo gene editing in the widely used C57BL/6 inbred strain.

MicroRNAs are small RNA species involved in biological control at multiple levels. Using genetic deletion and transgenic approaches, we show that the evolutionarily conserved microRNA-155 (miR-155) has an important role in the mammalian immune system, specifically in regulating T helper cell differentiation and the germinal center reaction to produce an optimal T cell-dependent antibody response. miR-155 exerts this control, at least in part, by regulating cytokine production. These results also suggest that individual microRNAs can exert critical control over mammalian differentiation processes in vivo.

The Ten-Eleven-Translocation 2 (TET2) gene encodes a member of TET family enzymes that alters the epigenetic status of DNA by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies, including myelodysplastic syndromes, myeloproliferative neoplasms, and chronic myelomonocytic leukemia. By analyzing mice with targeted disruption of the Tet2 catalytic domain, we show here that Tet2 is a critical regulator of self-renewal and differentiation of hematopoietic stem cells (HSCs). Tet2 deficiency led to decreased genomic levels of 5hmC and augmented the size of the hematopoietic stem/progenitor cell pool in a cell-autonomous manner. In competitive transplantation assays, Tet2-deficient HSCs were capable of multilineage reconstitution and possessed a competitive advantage over wild-type HSCs, resulting in enhanced hematopoiesis into both lymphoid and myeloid lineages. In vitro, Tet2 deficiency delayed HSC differentiation and skewed development toward the monocyte/macrophage lineage. Our data indicate that Tet2 has a critical role in regulating the expansion and function of HSCs, presumably by controlling 5hmC levels at genes important for the self-renewal, proliferation, and differentiation of HSCs.

A 1975 Nature paper reported how cell lines could be made that produce an antibody of known specificity. This discovery led to major biological insights and clinical successes in treating autoimmunity and cancer. Method to make antibodies reported in 1975 transformed science and medicine.

B2 cells engage in classical antibody responses, whereas B1 cells are considered carriers of innate immunity, biased toward recognizing epitopes present on the surfaces of common pathogens and self antigens. To explore the role of B cell antigen receptor (BCR) specificity in driving B1 cell differentiation, we developed a transgenic system allowing us to change BCR specificity in B cells in an inducible and programmed manner. Mature B2 cells differentiated into bona fide B1 cells upon acquisition of a B1 cell–typical self-reactive BCR through a phase of proliferative expansion. Thus, B2 cells have B1 cell differentiation potential in addition to their classical capacity to differentiate into memory and plasma cells, and B1 differentiation can be instructed by BCR-mediated self-reactivity, in the absence of B1-lineage precommitment.

Each antibody-producing B cell makes antibodies of unique specificity, reflecting a series of ordered gene rearrangements which must be successfully performed if the cell is to survive. A second selection process occurs during immune responses in which a new antibody repertoire is generated through somatic hypermutation. Here only mutants binding antigen with high affinity survive to become memory cells. Cells expressing autoreactive receptors are counter-selected at both stages. This stringent positive and negative selection allows the generation and diversification of cells while rigorously controlling their specificity.

The regulator c-Myc is well known for controlling cell growth but, paradoxically, evidence for its involvement in germinal centers has proven elusive. Rajewsky and colleagues show that it is essential for their development and maintenance. Germinal centers (GCs) are sites of intense B cell proliferation and are central for T cell–dependent antibody responses. However, the role of c-Myc, a key cell-cycle regulator, in this process has been questioned. Here we identified c-Myc + B cell subpopulations in immature and mature GCs and found, by genetic ablation of Myc , that they had indispensable roles in the formation and maintenance of GCs. The identification of these functionally critical cellular subsets has implications for human B cell lymphomagenesis, which originates mostly from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the c-Myc + GC subpopulations may be at a particularly high risk for malignant transformation.

Applying clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9)-mediated mutagenesis to primary mouse immune cells, we used high-fidelity single guide RNAs (sgRNAs) designed with an sgRNA design tool (CrispRGold) to target genes in primary B cells, T cells, and macrophages isolated from a Cas9 transgenic mouse line. Using this system, we achieved an average knockout efficiency of 80% in B cells. On this basis, we established a robust small-scale CRISPR-mediated screen in these cells and identified genes essential for B-cell activation and plasma cell differentiation. This screening system does not require deep sequencing and may serve as a precedent for the application of CRISPR/Cas9 to primary mouse cells.