Efficient CRISPR/Cas9-mediated gene knockin in mouse hematopoietic stem and progenitor cells

Mutations accumulating in hematopoietic stem and progenitor cells (HSPCs) during development can cause severe hematological disorders. Modeling these mutations in mice is essential for understanding their functional consequences. Here, we describe an efficient CRISPR/Cas9-based system to knock in and repair genes in mouse HSPCs. CRISPR/Cas9 ribonucleoproteins, in combination with recombinant adeno-associated virus (rAAV)-DJ donor templates, led to gene knockin efficiencies of up to 30% in the Lmnb1 and Actb loci of mouse HSPCs in vitro. The targeted HSPCs engraft and reconstitute all immune cell lineages in the recipient mice. Using this approach, we corrected a neomycin-disrupted Rag2 gene. The Rag2-corrected HSPCs restore B and T cell development in vivo, confirming the functionality of the approach. Our method provides an efficient strategy to study gene function in the hematopoietic system and model hematological disorders in vivo, without the need for germline mutagenesis

Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

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

Genome-Wide Analysis of leafbladeless1-Regulated and Phased Small RNAs Underscores the Importance of the TAS3 ta-siRNA Pathway to Maize Development

Maize leafbladeless1 (lbl1) encodes a key component in the trans-acting short-interfering RNA (ta-siRNA) biogenesis pathway. Correlated with a great diversity in ta-siRNAs and the targets they regulate, the phenotypes conditioned by mutants perturbing this small RNA pathway vary extensively across species. Mutations in lbl1 result in severe developmental defects, giving rise to plants with radial, abaxialized leaves. To investigate the basis for this phenotype, we compared the small RNA content between wild-type and lbl1 seedling apices. We show that LBL1 affects the accumulation of small RNAs in all major classes, and reveal unexpected crosstalk between ta-siRNA biogenesis and other small RNA pathways regulating transposons. Interestingly, in contrast to data from other plant species, we found no evidence for the existence of phased siRNAs generated via the one-hit model. Our analysis identified nine TAS loci, all belonging to the conserved TAS3 family. Information from RNA deep sequencing and PARE analyses identified the tasiR-ARFs as the major functional ta-siRNAs in the maize vegetative apex where they regulate expression of AUXIN RESPONSE FACTOR3 (ARF3) homologs. Plants expressing a tasiR-ARF insensitive arf3a transgene recapitulate the phenotype of lbl1, providing direct evidence that deregulation of ARF3 transcription factors underlies the developmental defects of maize ta-siRNA biogenesis mutants. The phenotypes of Arabidopsis and Medicago ta-siRNA mutants, while strikingly different, likewise result from misexpression of the tasiR-ARF target ARF3. Our data indicate that diversity in TAS pathways and their targets cannot fully account for the phenotypic differences conditioned by ta-siRNA biogenesis mutants across plant species. Instead, we propose that divergence in the gene networks downstream of the ARF3 transcription factors or the spatiotemporal pattern during leaf development in which these proteins act constitute key factors underlying the distinct contributions of the ta-siRNA pathway to development in maize, Arabidopsis, and possibly other plant species as well

Distribuição longitudinal de Chironomidae (Diptera) abaixo de uma barragem em um rio neotropical

The damming of a river causes dangerous consequences on structure of the environment downstream of the dam, modifying the sediment composition, which impose major adjustments in longitudinal distribution of benthic community. The construction of Engenheiro Sérgio Motta Dam in the Upper Paraná River has caused impacts on the aquatic communities, which are not yet fully known. This work aimed to provide more information about the effects of this impoundment on the structure of Chironomidae larvae assemblage. The analysis of data of physical and chemical variables in relation to biological data of 8 longitudinal sections in the Upper Paraná River showed that composition of Chironomidae larvae of stations near Engenheiro Sérgio Motta Dam differed of the other stations (farther of the Dam) The predominance of coarse sediments at stations upstream and finer sediments further downstream affected the choice of habitat by different morphotypes of Chironomidae and it caused a change in the structure of this assemblage in the longitudinal stretch.O barramento de um rio pode causar graves consequências sobre a natureza do ambiente, abaixo da barragem, modificando a composição do sedimento, as quais impõem importantes ajustes da distribuição longitudinal das comunidades bentônicas. A construção da Usina Hidrelétrica Engenheiro Sérgio Motta no alto rio Paraná, tem causado impactos em várias comunidades aquáticas, que ainda não são totalmente conhecidos. Este trabalho objetivou fornecer mais informações sobre os efeitos desse represamento na assembleia de Chironomidae. A análise das variáveis físicas e químicas em relação aos dados biológicos de oito transectos longitudinais no alto rio Paraná revelou que a composição das larvas de Chironomidae das estações mais próximas à barragem da Usina Engenheiro Sérgio Motta diferiu das demais (estações mais distantes). A predominância de sedimentos mais grosseiros nas estações a montante e sedimentos mais finos mais a jusante afetou a escolha de habitat pelos diferentes morfotipos de Chironomidae, que levou a alteração na estrutura desta assembleia ao longo do trecho amostrado.Fil: Pinha, G. D.. Universidade Estadual de Maringá. Programa de Pós-Graduação em Ecologia de Ambientes Aquáticos Continentais; Brasil.;Fil: Aviz, D.. Universidade Federal Do Pará; Brasil.;Fil: Lopes Filho, D. R.. Universidade Estadual de Maringá. Programa de Pós-Graduação em Ecologia de Ambientes Aquáticos Continentais; Brasil.;Fil: Petsch, D. K.. Universidade Estadual de Maringá. Programa de Pós-Graduação em Ecologia de Ambientes Aquáticos Continentais; Brasil.;Fil: Marchese Garello, Mercedes Rosa. Consejo Nacional de Investigaciones científicas y Técnicas. Centro Científico Tecnológico CONICET- Santa Fe. Instituto Nacional de Limnologia (i); Argentina;Fil: Takeda, A. M.. Universidade Estadual de Maringá; Brasil.

Adjuvant effects of a sequence-engineered mRNA vaccine: translational profiling demonstrates similar human and murine innate response

Additional file 1: Fig. S1. Components and assembly of the PTE module for innate responses. (A) Top panel—in vivo monocytes continuously emigrate from the blood into peripheral tissues with a half-life in the blood of ~1 day. Bottom panel: In vitro generation of cytokine derived DCs from monocytes involves the addition of IL-4 and GM-CSF and culture for 7–11 days. (B) Schematic of the components of a MIMIC-PTE module. In the 3D PTE, differentiation occurs in hours to about 2 days triggered by migration into and out of (reverse migration) through the endothelium: a process reminiscent of the movement of cells from tissues into lymphatic vessels

Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line

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

Mendelian and Non-Mendelian Regulation of Gene Expression in Maize

Transcriptome variation plays an important role in affecting the phenotype of an organism. However, an understanding of the underlying mechanisms regulating transcriptome variation in segregating populations is still largely unknown. We sought to assess and map variation in transcript abundance in maize shoot apices in the intermated B73×Mo17 recombinant inbred line population. RNA-based sequencing (RNA-seq) allowed for the detection and quantification of the transcript abundance derived from 28,603 genes. For a majority of these genes, the population mean, coefficient of variation, and segregation patterns could be predicted by the parental expression levels. Expression quantitative trait loci (eQTL) mapping identified 30,774 eQTL including 96 trans-eQTL "hotspots," each of which regulates the expression of a large number of genes. Interestingly, genes regulated by a trans-eQTL hotspot tend to be enriched for a specific function or act in the same genetic pathway. Also, genomic structural variation appeared to contribute to cis-regulation of gene expression. Besides genes showing Mendelian inheritance in the RIL population, we also found genes whose expression level and variation in the progeny could not be predicted based on parental difference, indicating that non-Mendelian factors also contribute to expression variation. Specifically, we found 145 genes that show patterns of expression reminiscent of paramutation such that all the progeny had expression levels similar to one of the two parents. Furthermore, we identified another 210 genes that exhibited unexpected patterns of transcript presence/absence. Many of these genes are likely to be gene fragments resulting from transposition, and the presence/absence of their transcripts could influence expression levels of their ancestral syntenic genes. Overall, our results contribute to the identification of novel expression patterns and broaden the understanding of transcriptional variation in plants. © 2013 Lin et al

Genic and nongenic contributions to natural variation of quantitative traits in maize

The complex genomes of many economically important crops present tremendous challenges to understand the genetic control of many quantitative traits with great importance in crop production, adaptation, and evolution. Advances in genomic technology need to be integrated with strategic genetic design and novel perspectives to break new ground. Complementary to individual-gene-targeted research, which remains challenging, a global assessment of the genomic distribution of trait-associated SNPs (TASs) discovered from genome scans of quantitative traits can provide insights into the genetic architecture and contribute to the design of future studies. Here we report the first systematic tabulation of the relative contribution of different genomic regions to quantitative trait variation in maize. We found that TASs were enriched in the nongenic regions, particularly within a 5-kb window upstream of genes, which highlights the importance of polymorphisms regulating gene expression in shaping the natural variation. Consistent with these findings, TASs collectively explained 44%-59% of the total phenotypic variation across maize quantitative traits, and on average, 79% of the explained variation could be attributed to TASs located in genes or within 5 kb upstream of genes, which together comprise only 13% of the genome. Our findings suggest that efficient, cost-effective genome-wide association studies (GWAS) in species with complex genomes can focus on genic and promoter regions