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 non-homologous 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 8-fold 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.The clustered, regularly interspaced, short palindromic repeats (CRISPRs)-Cas9 endonuclease represent a versatile tool for genome engineering, enabling the induction of site-specific genomic double-strand breaks (DSBs) by single guide RNAs (sgRNAs) 1 .In mammalian cells DSBs are mostly repaired by the non-homologous end joining (NHEJ) pathway 2,3 , frequently leading to the loss of nucleotides from the DSB ends. This enables the efficient construction of knockout alleles through the induction of frameshift To quantitatively determine the outcome of CRISPR-induced DSB repair, we first generated human HEK293 cells with a `traffic light´ reporter 7 (TLR) vector integrated into the AAVS1 locus 8 (Fig. 1a). HEK293 cells were transfected with an AAVS1 targeting vector carrying the TLR insert and Cas9 and AAVS1-specific sgRNA expression plasmids ( Supplementary Fig. 1 , and up to 7-fold by the Ad4 protein pair ( Fig. 2c and Supplementary Fig. 2.2).Titration of SCR7 on AAVS1 TLR/+ cells showed an optimal effect at 1 µM concentration ( Supplementary Fig. 2.3). In the presence of two shRNAs, SCR7 or Ad4 proteins we noticed diminished fluorescence signals within the population of Venus + cells at 72h after transfection (Fig. 2a, Supplementary Fig. 2.1, 2.3c), indicating reduced Venus expression in cells undergoing NHEJ blockade, possibly caused by local chromatin remodeling through an extended DNA damage response 13,14 . However, Venus expression was normal in clones established from AAVS1 TLR/+ cells targeted in the presence of Ad4 proteins, indicating that this effect is only transient (Supplementary Fig. 2.3d). From the sample expressing the Ad4 proteins, Venus + cells were sorted, and we established 24 clones to confirm the integrity of the repaired TLR loci using PCR and sequence analysis (Supplementary Table 2). In contrast to the increase of Venus + cells, RFP + cells decreased from 3% in the controls to 1.7%, 1.4% or 0.6% in the presence of 5 shRNAs, SCR7 or Ad4 proteins, respectively (Fig. 2a, 2b). Whether the residual NHEJ activity relies on the KU/Ligase IV independent alternative end-joining mechanism 15 rem...

Plasma cells are of crucial importance for long-term immune protection. It is thought that long-lived plasma cells survive in specialized niches in the bone marrow. Here we demonstrate that bone marrow eosinophils localized together with plasma cells and were the key providers of plasma cell survival factors. In vitro, eosinophils supported the survival of plasma cells by secreting the proliferation-inducing ligand APRIL and interleukin-6 (IL-6). In eosinophil-deficient mice, plasma cell numbers were much lower in the bone marrow both at steady state and after immunization. Reconstitution experiments showed that eosinophils were crucial for the retention of plasma cells in the bone marrow. Moreover, depletion of eosinophils induced apoptosis in long-lived bone marrow plasma cells. Our findings demonstrate that the long-term maintenance of plasma cells in the bone marrow requires eosinophils.

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).

Although in normal lamina propria (LP) large numbers of eosinophils are present, little is known about their role in mucosal immunity at steady state. Here we show that eosinophils are needed to maintain immune homeostasis in gut-associated tissues. By using eosinophil-deficient ΔdblGATA-1 and PHIL mice or an eosinophil-specific depletion model, we found a reduction in immunoglobulin A(+) (IgA(+)) plasma cell numbers and in secreted IgA. Eosinophil-deficient mice also showed defects in the intestinal mucous shield and alterations in microbiota composition in the gut lumen. In addition, TGF-β-dependent events including class switching to IgA in Peyer's patches (PP), the formation of CD103(+) T cells including Foxp3(+) regulatory (Treg), and also CD103(+) dendritic cells were disturbed. In vitro cultures showed that eosinophils produce factors that promote T-independent IgA class switching. Our findings show that eosinophils are important players for immune homeostasis in gut-associated tissues and add to data suggesting that eosinophils can promote tissue integrity.