scholarly journals Mappingin vivogenetic interactomics through Cpf1 crRNA array screening

2017 ◽  
Author(s):  
Ryan D. Chow ◽  
Guangchuan Wang ◽  
Adan Codina ◽  
Lupeng Ye ◽  
Sidi Chen
Keyword(s):  
Genome Editing ◽  
Biological Networks ◽  
Mammalian Species ◽  
Simple Approach ◽  
Genetic Interactions ◽  
Epigenetic Factors ◽  
Double Knockout ◽  
Mammalian Genomes ◽  
Single Effector

AbstractGenetic interactions lay the foundation of biological networks in virtually all organisms. Due to the complexity of mammalian genomes and cellular architectures, unbiased mapping of genetic interactionsin vivois challenging. Cpf1 is a single effector RNA-guided nuclease that enables multiplexed genome editing using crRNA arrays. Here we designed a Cpf1 crRNA array library targeting all pairwise permutations of the most significantly mutated nononcogenes, and performed double knockout screens in mice using a model of malignant transformation as well as a model of metastasis. CrRNA array sequencing revealed a quantitative landscape of all single and double knockouts. Enrichment, synergy and clonal analyses identified many unpredicted drivers and co-drivers of transformation and metastasis, with epigenetic factors as hubs of these highly connected networks. Our study demonstrates a powerful yet simple approach forin vivomapping of unbiased genetic interactomes in mammalian species at a phenotypic level.

scholarly journals Welcoming a new age for gene therapy in hematology

Blood ◽  
2016 ◽  
Vol 127 (21) ◽  
pp. 2523-2524 ◽  
Author(s):  
Mitchell J. Weiss ◽  
Charles G. Mullighan
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Genome Editing ◽  
Gene Targeting ◽  
Embryonic Stem ◽  
Hematological Disorders ◽  
Cellular Genes ◽  
Mammalian Genomes ◽  
Template Dna

Abstract Our capacities to understand and manipulate mammalian genomes are accelerating at an astounding pace. In 2007, Capecchi, Evans, and Smithies were awarded the Nobel Prize in medicine for their work on gene targeting, which showed that embryonic stem cells could be modified by homologous recombination (HR) with engineered template DNA to alter virtually any gene and create mutant mice. This work revolutionized biology by allowing investigators to study the in vivo consequences of selected gene alteration. However, the efficiency of HR in embryonic stem cells is unpredictable, depending on the target gene and HR template. More importantly, spontaneous HR occurs at very low rates in most somatic cells, restricting the use of standard gene targeting for most laboratory and clinical applications. This limitation is being overcome by genome-editing technologies, which markedly enhance the capacity to alter cellular genes with laser-like precision. Four review articles in this edition of Blood summarize the field of genome editing, focusing on its potential for treating hematological disorders.

scholarly journals Find and cut-and-transfer (FiCAT) mammalian genome engineering

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Maria Pallarès-Masmitjà ◽  
Dimitrije Ivančić ◽  
Júlia Mir-Pedrol ◽  
Jessica Jaraba-Wallace ◽  
Tommaso Tagliani ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Genome Editing ◽  
Mouse Liver ◽  
Genome Engineering ◽  
Mammalian Genome ◽  
Transfer Efficiency ◽  
Dna Fragments ◽  
Efficient Delivery ◽  
Mammalian Genomes

AbstractWhile multiple technologies for small allele genome editing exist, robust technologies for targeted integration of large DNA fragments in mammalian genomes are still missing. Here we develop a gene delivery tool (FiCAT) combining the precision of a CRISPR-Cas9 (find module), and the payload transfer efficiency of an engineered piggyBac transposase (cut-and-transfer module). FiCAT combines the functionality of Cas9 DNA scanning and targeting DNA, with piggyBac donor DNA processing and transfer capacity. PiggyBac functional domains are engineered providing increased on-target integration while reducing off-target events. We demonstrate efficient delivery and programmable insertion of small and large payloads in cellulo (human (Hek293T, K-562) and mouse (C2C12)) and in vivo in mouse liver. Finally, we evolve more efficient versions of FiCAT by generating a targeted diversity of 394,000 variants and undergoing 4 rounds of evolution. In this work, we develop a precise and efficient targeted insertion of multi kilobase DNA fragments in mammalian genomes.

A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription

2020 ◽  
Vol 42 (7) ◽  
pp. 1203-1210 ◽  
Author(s):  
Chuan Wu ◽  
Yumeng Chen ◽  
Yifei Qiu ◽  
Xiao Niu ◽  
Ningjian Zhu ◽  
...  
Keyword(s):  
Genome Editing ◽  
Trichoderma Reesei ◽  
Filamentous Fungus ◽  
Simple Approach

scholarly journals Novel Role for Albumin in Innate Immunity: Serum Albumin Inhibits the Growth of Blastomyces dermatitidis Yeast Form In Vitro

2003 ◽  
Vol 71 (11) ◽  
pp. 6648-6652 ◽  
Author(s):  
Steven Giles ◽  
Charles Czuprynski
Keyword(s):  
Molecular Weight ◽  
Inhibitory Activity ◽  
Mammalian Species ◽  
Blastomyces Dermatitidis ◽  
Low Molecular Weight ◽  
Rat Serum ◽  
Yeast Form ◽  
Domain Iii

ABSTRACT In this study we found that serum inhibitory activity against Blastomyces dermatitidis was principally mediated by albumin. This was confirmed in experiments using albumin from several mammalian species. Analbuminemic rat serum did not inhibit B. dermatitidis growth in vivo; however, the addition of albumin restored inhibitory activity. Inhibitory activity does not require albumin domain III and appears to involve binding of a low-molecular-weight yeast-derived growth factor.

scholarly journals A Set of Cell Lines Derived from a Genetic Murine Glioblastoma Model Recapitulates Molecular and Morphological Characteristics of Human Tumors

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 230
Author(s):  
Barbara Costa ◽  
Michael N.C. Fletcher ◽  
Pavle Boskovic ◽  
Ekaterina L. Ivanova ◽  
Tanja Eisemann ◽  
...  
Keyword(s):  
Cell Lines ◽  
Immune Cell ◽  
Treatment Options ◽  
Morphological Characteristics ◽  
Molecular Heterogeneity ◽  
Double Knockout ◽  
In Vivo Models ◽  
Human Glioblastoma ◽  
Human Gbm

Glioblastomas (GBM) are the most aggressive tumors affecting the central nervous system in adults, causing death within, on average, 15 months after diagnosis. Immunocompetent in-vivo models that closely mirror human GBM are urgently needed for deciphering glioma biology and for the development of effective treatment options. The murine GBM cell lines currently available for engraftment in immunocompetent mice are not only exiguous but also inadequate in representing prominent characteristics of human GBM such as infiltrative behavior, necrotic areas, and pronounced tumor heterogeneity. Therefore, we generated a set of glioblastoma cell lines by repeated in vivo passaging of cells isolated from a neural stem cell-specific Pten/p53 double-knockout genetic mouse brain tumor model. Transcriptome and genome analyses of the cell lines revealed molecular heterogeneity comparable to that observed in human glioblastoma. Upon orthotopic transplantation into syngeneic hosts, they formed high-grade gliomas that faithfully recapitulated the histopathological features, invasiveness and immune cell infiltration characteristic of human glioblastoma. These features make our cell lines unique and useful tools to study multiple aspects of glioblastoma pathomechanism and to test novel treatments in an intact immune microenvironment.

scholarly journals Aptardi predicts polyadenylation sites in sample-specific transcriptomes using high-throughput RNA sequencing and DNA sequence

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ryan Lusk ◽  
Evan Stene ◽  
Farnoush Banaei-Kashani ◽  
Boris Tabakoff ◽  
Katerina Kechris ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Dna Sequence ◽  
Mammalian Species ◽  
Alternative Polyadenylation ◽  
Sequence Information ◽  
Rna Seq ◽  
Average Precision ◽  
Polyadenylation Sites ◽  
Dna Nucleotide Sequence

AbstractAnnotation of polyadenylation sites from short-read RNA sequencing alone is a challenging computational task. Other algorithms rooted in DNA sequence predict potential polyadenylation sites; however, in vivo expression of a particular site varies based on a myriad of conditions. Here, we introduce aptardi (alternative polyadenylation transcriptome analysis from RNA-Seq data and DNA sequence information), which leverages both DNA sequence and RNA sequencing in a machine learning paradigm to predict expressed polyadenylation sites. Specifically, as input aptardi takes DNA nucleotide sequence, genome-aligned RNA-Seq data, and an initial transcriptome. The program evaluates these initial transcripts to identify expressed polyadenylation sites in the biological sample and refines transcript 3′-ends accordingly. The average precision of the aptardi model is twice that of a standard transcriptome assembler. In particular, the recall of the aptardi model (the proportion of true polyadenylation sites detected by the algorithm) is improved by over three-fold. Also, the model—trained using the Human Brain Reference RNA commercial standard—performs well when applied to RNA-sequencing samples from different tissues and different mammalian species. Finally, aptardi’s input is simple to compile and its output is easily amenable to downstream analyses such as quantitation and differential expression.

Sign in / Sign up

Export Citation Format

Share Document