Browsing by Subject "CRISPR"

Now showing 1 - 10 of 14
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    Author Correction: CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity
    (Nature Publishing Group, 2018-04-13) Walton, Josephine B.; Farquharson, Malcolm; Mason, Susan; Port, Jennifer; Kruspig, Bjorn; Dowson, Suzanne; Stevenson, David; Murphy, Daniel; Matzuk, Martin; Kim, Jaeyeon; Coffelt, Seth; Blyth, Karen; McNeish, Iain A.; Biochemistry and Molecular Biology, School of Medicine
    A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
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    CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion
    (BMC, 2022) Krohannon, Alexander; Srivastava, Mansi; Rauch, Simone; Srivastava, Rajneesh; Dickinson, Bryan C.; Janga, Sarath Chandra; BioHealth Informatics, School of Informatics and Computing
    Background: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited. Results: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. Conclusions: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.
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    CRISPR-Cas9 Mediated Epitope Tagging Provides Accurate and Versatile Assessment of Myocardin
    (American Heart Association, 2018-09) Lyu, Qing; Dhagia, Vidhi; Han, Yu; Guo, Bing; Wines-Samuelson, Mary E.; Christie, Christine K.; Yin, Qiangzong; Slivano, Orazio J.; Herring, Paul; Long, Xiaochun; Gupte, Sachin A.; Miano, Joseph M.; Cellular and Integrative Physiology, School of Medicine
    Objective- Unreliable antibodies often hinder the accurate detection of an endogenous protein, and this is particularly true for the cardiac and smooth muscle cofactor, MYOCD (myocardin). Accordingly, the mouse Myocd locus was targeted with 2 independent epitope tags for the unambiguous expression, localization, and activity of MYOCD protein. Approach and Results- 3cCRISPR (3-component clustered regularly interspaced short palindromic repeat) was used to engineer a carboxyl-terminal 3×FLAG or 3×HA epitope tag in mouse embryos. Western blotting with antibodies to each tag revealed a MYOCD protein product of ≈150 kDa, a size considerably larger than that reported in virtually all publications. MYOCD protein was most abundant in some adult smooth muscle-containing tissues with surprisingly low-level expression in the heart. Both alleles of Myocd are active in aorta because a 2-fold increase in protein was seen in mice homozygous versus heterozygous for FLAG-tagged Myocd. ChIP (chromatin immunoprecipitation)-quantitative polymerase chain reaction studies provide proof-of-principle data demonstrating the utility of this mouse line in conducting genome-wide ChIP-seq studies to ascertain the full complement of MYOCD-dependent target genes in vivo. Although FLAG-tagged MYOCD protein was undetectable in sections of adult mouse tissues, low-passaged vascular smooth muscle cells exhibited expected nuclear localization. Conclusions- This report validates new mouse models for analyzing MYOCD protein expression, localization, and binding activity in vivo and highlights the need for rigorous authentication of antibodies in biomedical research.
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    CRISPR-Cas9-mediated insertion of a short artificial intron for the generation of conditional alleles in mice
    (Elsevier, 2023) Cassidy, Annelise; Pelletier, Stephane; Medical and Molecular Genetics, School of Medicine
    In this protocol, we describe the generation of conditional alleles in mice using the DECAI (DEgradation based on Cre-regulated Artificial Intron) approach. We detail steps for the CRISPR-mediated insertion of the short DECAI cassette within exon 3 of Scyl1 and the functional validation of alleles at genomic, transcriptomic, and protein levels. This strategy simplifies the process of generating mice with conditional alleles.
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    CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity
    (Nature Publishing group, 2017-12-04) Walton, Josephine B.; Farquharson, Malcolm; Mason, Susan; Port, Jennifer; Kruspig, Bjorn; Dowson, Suzanne; Stevenson, David; Murphy, Daniel; Matzuk, Martin; Kim, Jaeyeon; Coffelt, Seth; Blyth, Karen; McNeish, Iain A.; Biochemistry and Molecular Biology, School of Medicine
    Transplantable murine models of ovarian high grade serous carcinoma (HGSC) remain an important research tool. We previously showed that ID8, a widely-used syngeneic model of ovarian cancer, lacked any of the frequent mutations in HGSC, and used CRISPR/Cas9 gene editing to generate derivatives with deletions in Trp53 and Brca2. Here we have used one ID8 Trp53 −/− clone to generate further mutants, with additional mutations in Brca1, Pten and Nf1, all of which are frequently mutated or deleted in HGSC. We have also generated clones with triple deletions in Trp53, Brca2 and Pten. We show that ID8 Trp53 −/−;Brca1 −/− and Trp53 −/−;Brca2 −/− cells have defective homologous recombination and increased sensitivity to both platinum and PARP inhibitor chemotherapy compared to Trp53 −/−. By contrast, loss of Pten or Nf1 increases growth rate in vivo, and reduces survival following cisplatin chemotherapy in vivo. Finally, we have also targeted Trp53 in cells isolated from a previous transgenic murine fallopian tube carcinoma model, and confirmed that loss of p53 expression in this second model accelerates intraperitoneal growth. Together, these CRISPR-generated models represent a new and simple tool to investigate the biology of HGSC, and the ID8 cell lines are freely available to researchers.
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    Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes
    (Wiley, 2015-05) Estrada, J.; Martens, G.; Li, P.; Adams, A.B.; Newell, K.A.; Ford, M.L.; Butler, J.R.; Sidner, R.A.; Tector, M.; Tector, A.J.; Department of Surgery, IU School of Medicine
    Background Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes. Methods The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/β4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons. Results Cells from GGTA1/CMAH/β4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Nonhuman primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased nonhuman primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/β4GalNT2. Conclusions Inactivation of the β4GalNT2 gene reduces human and nonhuman primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of nonhuman primates towards GGTA1/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/β4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the nonhuman primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/β4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.
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    Generation of Casp8FL122/123GG Mice Using CRISPR-Cas9 Technology
    (Cell Press, 2020-12-18) Pelletier, Stephane; Tummers, Bart; Green, Douglas R.; Medical and Molecular Genetics, School of Medicine
    The purpose of this protocol is to describe the generation of missense mutations in mice using CRISPR-Cas9 technology. The current protocol focuses on the generation of a Casp8 FL122/123GG missense mutation, but it can be adapted to introduce any missense or nonsense mutation. For complete details on the use and execution of this protocol, please refer to Tummers et al. (2020).
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    Novel methods for the generation of genetically engineered animal models
    (Elsevier, 2023) Cassidy, Annelise; Onal, Melda; Pelletier, Stephane; Medical and Molecular Genetics, School of Medicine
    Genetically modified mouse models have shaped our understanding of biological systems in both physiological and pathological conditions. For decades, mouse genome engineering has relied on transgenesis and spontaneous gene replacement in embryonic stem (ES) cells. While these technologies provided a wealth of knowledge, they remain imprecise and expensive to use. Recent advances in genome editing technologies such as the development of targetable nucleases, the improvement of delivery systems, and the simplification of targeting strategies now allow for the rapid, precise manipulation of the mouse genome. In this review article, we discuss novel methods and targeting strategies for the generation of mouse models for the study of bone and skeletal muscle biology.
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    Nuclease-based editing in the porcine genome : a strategy to facilitate porcine-to human xenotransplantation
    (2017-04-18) Butler, James R.; Tector, A. Joseph; White, Kenneth E.; Schmidt, C. Max; Radovich, Milan
    Solid organ transplantation is severely limited by a shortage of available donor allografts. Pig-to-human xenotransplantation offers a potential solution to this growing problem. For xenotransplantation to achieve clinical relevance, both immunologic and physiologic barriers must be understood. Genetic modification of pigs has proven to be a valuable means of both studying and eliminating these barriers. The present body of work describes a method for greatly increasing the efficiency and precision of genome editing within the porcine genome. By combining non-integrating selection and homologous recombination of exogenous oligonucleotides, a method for rapidly creating genetic modification without reliance on phenotypic sorting was achieved. Furthermore this work employs the technique of CRISPR/Cas9-directed mutagenesis to create and analyze several new animal models of porcine-to-human xenotransplantation with respect to both immunologic and physiologic parameters. First, Isoglobotrihexosylceramide -a controversial glycan to the field of xenotransplantation- was studied in a knockout model and found not to affect human-anti-porcine humoral reactions. Second, a new combination of glycan modifications is described that significantly lowers the human anti-porcine humoral immune response. This model animal suggests that glycan-deletion alone will be sufficient to promote clinical application, and that conventional immunosuppression will be successful in mediating the human cellular response. Finally, two potential physiologic barriers to xenotransplantation are studied in genetically modified model animals. Xenogenic consumption of human platelets was studied across hepatic and renal organ systems; xenogenic platelet consumption was reduced by glycan modifications to the porcine liver while human platelet sequestration was not identified in the study of renal endothelium. Porcine FcRN –an essential receptor expressed in kidneys to maintain serum proteostasis- was studied as a final potential barrier to pig-to human renal transplantation. Because albumin is the primary driver of serum oncotic pressure, the protein-protein interaction of endogenous porcine FcRN and human albumin was studied. Porcine FcRN was found capable of binding human albumin under physiologic parameters. In summary, the results of the present work suggest that the salient barriers to clinical xenotransplantation have been removed and that porcine-to human renal transplantation may soon offer an answer to the current organ shortage.
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    Protocol for genome-scale in vivo CRISPR screening to study protection of beta cells under autoimmunity in a type 1 diabetes mouse model
    (Elsevier, 2023) Li, Jian; Lee, Yu-Chi; Iessi, Isabela L.; Wu, Chialing; Yi, Peng; Cai, Erica P.; Medicine, School of Medicine
    Autoimmunity-induced pancreatic beta cell failure is the main characteristic of type 1 diabetes (T1D). Here, we describe a protocol for genome-scale in vivo CRISPR-Cas9 screening for use in a mouse model of T1D. Using a non-obese-diabetic-derived mouse beta cell line, NIT-1, and a genome-wide CRISPR-Cas9 knockout library (GeCKO-v2), we describe how to identify genes that confer resistance to autoimmune killing. This protocol can be applied in other mouse models of autoimmunity. For complete details on the use and execution of this protocol, please refer to Cai et al. (2020).