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.
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IJS, NUK, SBMB, UL, UM, UPUK
Cas9 nucleases can be programmed with single guide RNAs (sgRNAs) to mediate gene editing. High CRISPR/Cas9-mediated gene knockout efficiencies are essential for genetic screens and critically depend ...on the properties of the sgRNAs used. The specificity of an sgRNA is defined by its targeting sequence. Here, we discovered that two short sequence motifs at the 3′ end of the targeting sequence are almost exclusively present in inefficient sgRNAs of published sgRNA-activity datasets. By specific knock-in of sgRNA target sequences with or without these motifs and quantitative measurement of knockout efficiency, we show that the presence of these motifs in sgRNAs per se results in a 10-fold reduction of gene knockout frequencies. Mechanistically, the cause of the low efficiency differs between the two motifs. These sequence motifs are relevant for future sgRNA design approaches and studies of Cas9-DNA interactions.
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•TT- and GCC-motifs are enriched in inefficient sgRNAs in published sgRNA-activity datasets•These sequence motifs are located in the 4 PAM-proximal bases of the targeting sequence•Their presence is sufficient to block CRISPR/Cas9-mediated gene editing•A modified scaffold RNA neutralizes the negative impact of the TT-motif
CRISPR/Cas9-mediated gene editing efficiency critically depends on the properties of the sgRNAs used. Graf et al. show that the presence of two position-specific sequence motifs in the targeting sequence of sgRNAs per se results in a severe reduction of gene knockout frequencies.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Germinal centers (GCs) are generally considered to be the sole site of memory B‐cell generation. However, recent studies demonstrate that memory B cells can also develop in response to a T‐cell ...dependent (TD) antigen before the onset, and independently of, the GC reaction. These two classes of memory cells persist equally over long periods of time and attain functional maturation through distinct but related transcriptional programs. Although the development of both memory B‐cell types requires classical T‐cell help, the generation of GC‐dependent memory B cells requires TFH‐cell help, while the generation of GC‐independent memory cells does not. These findings led to the conclusion that B‐cell memory is generated along two fundamentally distinct cellular differentiation pathways. In this review, we focus on the GC‐independent pathway of memory B‐cell development, and discuss how the unique features of memory B cells are maintained in the GC‐independent pathway.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
MiR-150 is a microRNA (miRNA) specifically expressed in mature lymphocytes, but not their progenitors. A top predicted target of miR-150 is c-Myb, a transcription factor controlling multiple steps of ...lymphocyte development. Combining loss- and gain-of-function gene targeting approaches for miR-150 with conditional and partial ablation of c-Myb, we show that miR-150 indeed controls c-Myb expression in vivo in a dose-dependent manner over a narrow range of miRNA and c-Myb concentrations and that this dramatically affects lymphocyte development and response. Our results identify a key transcription factor as a critical target of a stage-specifically expressed miRNA in lymphocytes and suggest that this and perhaps other miRNAs have evolved to control the expression of just a few critical target proteins in particular cellular contexts.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Summary
DNA methylation has pivotal regulatory roles in mammalian development, retrotransposon silencing, genomic imprinting, and X‐chromosome inactivation. Cancer cells display highly dysregulated ...DNA methylation profiles characterized by global hypomethylation in conjunction with hypermethylation of promoter CpG islands that presumably lead to genome instability and aberrant expression of tumor suppressor genes or oncogenes. The recent discovery of ten‐eleven‐translocation (TET) family dioxygenases that oxidize 5mC to 5‐hydroxymethylcytosine (5hmC), 5‐formylcytosine (5fC), and 5‐carboxylcytosine (5caC) in DNA has led to profound progress in understanding the mechanism underlying DNA demethylation. Among the three TET genes, TET2 recurrently undergoes inactivating mutations in a wide range of myeloid and lymphoid malignancies. TET2 functions as a bona fide tumor suppressor particularly in the pathogenesis of myeloid malignancies resembling chronic myelomonocytic leukemia (CMML) and myelodysplastic syndromes (MDS) in human. Here we review diverse functions of TET proteins and the novel epigenetic marks that they generate in DNA methylation/demethylation dynamics and normal and malignant hematopoietic differentiation. The impact of TET2 inactivation in hematopoiesis and various mechanisms modulating the expression or activity of TET proteins are also discussed. Furthermore, we also present evidence that TET2 and TET3 collaborate to suppress aberrant hematopoiesis and hematopoietic transformation. A detailed understanding of the normal and pathological functions of TET proteins may provide new avenues to develop novel epigenetic therapies for treating hematological malignancies.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Phosphatidylinositol 3′ OH kinase (PI3K) signaling and FOXO transcription factors play opposing roles at several B cell developmental stages. We show here abundant nuclear FOXO1 expression in the ...proliferative compartment of the germinal center (GC), its dark zone (DZ), and PI3K activity, downregulating FOXO1, in the light zone (LZ), where cells are selected for further differentiation. In the LZ, however, FOXO1 was expressed in a fraction of cells destined for DZ reentry. Upon FOXO1 ablation or induction of PI3K activity, GCs lost their DZ, owing at least partly to downregulation of the chemokine receptor CXCR4. Although this prevented proper cyclic selection of cells in GCs, somatic hypermutation and proliferation were maintained. Class switch recombination was partly lost due to a failure of switch region targeting by activation-induced deaminase (AID).
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•PI3K signaling and FOXO1 are antagonistic master regulators of the GC reaction•FOXO1 is essential for the proliferating GC compartment, the dark zone•Selection, but not generation, of somatic antibody mutants is controlled by FOXO1•FOXO1 controls isotype switching in GCs through switch region accessibility for AID
The germinal center (GC) reaction is of key importance for adaptive immunity as well as the origin of most human B cell lymphomas. Using conditional targeted mutagenesis, Sander, Rajewsky, and colleagues identify PI3K signaling and the transcription factor FOXO1 as essential antagonistic regulators of GC polarization, antibody diversification, and cellular selection.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
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.
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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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