Browsing by Subject "CRISPR/Cas9"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Creating class I MHC-null pigs using guide RNA and the Cas9 endonuclease
    (American Association of Immunologists, 2014-12-01) Reyes, Luz M.; Estrada, Jose L; Wang, Zheng Yu; Blosser, Rachel J.; Smith, Rashod F.; Sidner, Richard A.; Paris, Leela L.; Blankenship, Ross L.; Ray, Caitlin N.; Miner, Aaron C.; Tector, Matthew; Tector, A. Joseph; Surgery, School of Medicine
    Pigs are emerging as important large animal models for biomedical research, and they may represent a source of organs for xenotransplantation. The MHC is pivotal to the function of the immune system in health and disease, and it is particularly important in infection and transplant rejection. Pigs deficient in class I MHC could serve as important reagents to study viral immunity as well as allograft and xenograft rejection. In this study, we report the creation and characterization of class I MHC knockout pigs using the Cas9 nuclease and guide RNAs. Pig fetal fibroblasts were genetically engineered using Cas9 and guide RNAs, and class I MHC(-) cells were then used as nuclear donors for somatic cell nuclear transfer. We produced three piglets devoid of all cell surface class I proteins. Although these animals have reduced levels of CD4(-)CD8(+) T cells in peripheral blood, the pigs appear healthy and are developing normally. These pigs are a promising reagent for immunological research.
  • Thumbnail Image
    Item
    SliceIt: A genome-wide resource and visualization tool to design CRISPR/Cas9 screens for editing protein-RNA interaction sites in the human genome
    (Elsevier, 2020-06) Vemuri, Sasank; Srivastava, Rajneesh; Mir, Quoseena; Hashemikhabir, Seyedsasan; Dong, X. Charlie; Janga, Sarath Chandra; BioHealth Informatics, School of Informatics and Computing
    Several protein-RNA cross linking protocols have been established in recent years to delineate the molecular interaction of an RNA Binding Protein (RBP) and its target RNAs. However, functional dissection of the role of the RBP binding sites in modulating the post-transcriptional fate of the target RNA remains challenging. CRISPR/Cas9 genome editing system is being commonly employed to perturb both coding and noncoding regions in the genome. With the advancements in genome-scale CRISPR/Cas9 screens, it is now possible to not only perturb specific binding sites but also probe the global impact of protein-RNA interaction sites across cell types. Here, we present SliceIt (http://sliceit.soic.iupui.edu/), a database of in silico sgRNA (single guide RNA) library to facilitate conducting such high throughput screens. SliceIt comprises of ~4.8 million unique sgRNAs with an estimated range of 2-8 sgRNAs designed per RBP binding site, for eCLIP experiments of >100 RBPs in HepG2 and K562 cell lines from the ENCODE project. SliceIt provides a user friendly environment, developed using advanced search engine framework, Elasticsearch. It is available in both table and genome browser views facilitating the easy navigation of RBP binding sites, designed sgRNAs, exon expression levels across 53 human tissues along with prevalence of SNPs and GWAS hits on binding sites. Exon expression profiles enable examination of locus specific changes proximal to the binding sites. Users can also upload custom tracks of various file formats directly onto genome browser, to navigate additional genomic features in the genome and compare with other types of omics profiles. All the binding site-centric information is dynamically accessible via "search by gene", "search by coordinates" and "search by RBP" options and readily available to download. Validation of the sgRNA library in SliceIt was performed by selecting RBP binding sites in Lipt1 gene and designing sgRNAs. Effect of CRISPR/Cas9 perturbations on the selected binding sites in HepG2 cell line, was confirmed based on altered proximal exon expression levels using qPCR, further supporting the utility of the resource to design experiments for perturbing protein-RNA interaction networks. Thus, SliceIt provides a one-stop repertoire of guide RNA library to perturb RBP binding sites, along with several layers of functional information to design both low and high throughput CRISPR/Cas9 screens, for studying the phenotypes and diseases associated with RBP binding sites.
  • Thumbnail Image
    Item
    The Trem2 R47H variant confers loss-of-function-like phenotypes in Alzheimer's disease
    (BMC, 2018-06-01) Cheng-Hathaway, Paul J.; Reed-Geaghan, Erin G.; Jay, Taylor R.; Casali, Brad T.; Bemiller, Shane M.; Puntambekar, Shweta S.; von Saucken, Victoria E.; Williams, Roxanne Y.; Karlo, J. Colleen; Moutinho, Miguel; Xu, Guixiang; Ransohoff, Richard M.; Lamb, Bruce T.; Landreth, Gary E.; Anatomy and Cell Biology, School of Medicine
    BACKGROUND: The R47H variant of Triggering Receptor Expressed on Myeloid cells 2 (TREM2) confers greatly increased risk for Alzheimer's disease (AD), reflective of a central role for myeloid cells in neurodegeneration. Understanding how this variant confers AD risk promises to provide important insights into how myeloid cells contribute to AD pathogenesis and progression. METHODS: In order to investigate this mechanism, CRISPR/Cas9 was used to generate a mouse model of AD harboring one copy of the single nucleotide polymorphism (SNP) encoding the R47H variant in murine Trem2. TREM2 expression, myeloid cell responses to amyloid deposition, plaque burden, and neuritic dystrophy were assessed at 4 months of age. RESULTS: AD mice heterozygous for the Trem2 R47H allele exhibited reduced total Trem2 mRNA expression, reduced TREM2 expression around plaques, and reduced association of myeloid cells with plaques. These results were comparable to AD mice lacking one copy of Trem2. AD mice heterozygous for the Trem2 R47H allele also showed reduced myeloid cell responses to amyloid deposition, including a reduction in proliferation and a reduction in CD45 expression around plaques. Expression of the Trem2 R47H variant also reduced dense core plaque number but increased plaque-associated neuritic dystrophy. CONCLUSIONS: These data suggest that the AD-associated TREM2 R47H variant increases risk for AD by conferring a loss of TREM2 function and enhancing neuritic dystrophy around plaques.
  • Thumbnail Image
    Item
    Utility of CRISPR/Cas9 systems in hematology research
    (Elsevier, 2017) Lucas, Daniel; O'Leary, Heather A.; Ebert, Benjamin L.; Cowan, Chad A.; Tremblay, Cedric S.; Department of Microbiology and Immunology, IU School of Medicine
    Since the end of the 20th century, the development of novel approaches have emerged to manipulate experimental models of hematological disorders, so they would more accurately mirror what is observed in the clinic. Despite these technological advances, the characterization of crucial genes for benign or malignant hematological disorders remains challenging, mainly because of the dynamic nature of the hematopoietic system and the genetic heterogeneity of these disorders. To overcome this limitation, genome editing technologies have been developed to specifically manipulate the genome via deletion, insertion or modification of targeted loci. These technologies have swiftly progressed, allowing their common use to investigate genetic function in experimental hematology. Amongst them, homologous recombination (HR)-mediated targeting technologies have facilitated the manipulation of specific loci by generating knockout and knock-in models. Despite promoting significant advances in the understanding of the molecular mechanisms involved in hematology, these inefficient, time-consuming and labor-intensive approaches did not permit the development of cellular or animal models recapitulating the complexity of hematological disorders. In October 2016, Dr. Ben Ebert and Dr. Chad Cowan shared their knowledge and experiences with the utilization of CRISPR for models of myeloid malignancy, disease, and novel therapeutics. Here we provide an overview of the topics they covered including insights into the novel applications of the technique as well as its strengths and limitations.