CRISPR-Cas9 Mediated Gene-Silencing of the Mutant Huntingtin Gene in an In Vitro Model of Huntington's Disease.
Kolli Nivya,Lu Ming,Maiti Panchanan,Rossignol Julien,Dunbar Gray L
International journal of molecular sciences
Huntington's disease (HD) is a fatal neurodegenerative genetic disease characterized by a loss of neurons in the striatum. It is caused by a mutation in the Huntingtin gene () that codes for the protein huntingtin (HTT). The mutant Huntingtin gene (m) contains extra poly-glutamine (CAG) repeats from which the translated mutant huntingtin proteins (mHTT) undergo inappropriate post-translational modifications, conferring a toxic gain of function, in addition to its non-functional property. In order to curb the production of the mHTT, we have constructed two CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR associate protein) plasmids, among which one nicks the DNA at untranslated region upstream to the open reading frame (uORF), and the other nicks the DNA at exon1-intron boundary. The primary goal of this study was to apply this plasmid into mesenchymal stem cells (MSCs) extracted from the bone-marrow of YAC128 mice, which carries the transgene for HD. Our results suggest that the disruption of uORF through CRISPR-Cas9 influences the translation of mHTT negatively and, to a lesser extent, disrupts the exon1-intron boundary, which affects the translation of the mHTT. These findings also revealed the pattern of the nucleotide addition or deletion at the site of the DNA-nick in this model.
One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.
Wang Haoyi,Yang Hui,Shivalila Chikdu S,Dawlaty Meelad M,Cheng Albert W,Zhang Feng,Jaenisch Rudolf
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty--8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
A neuroscientist's guide to transgenic mice and other genetic tools.
Navabpour Shaghayegh,Kwapis Janine L,Jarome Timothy J
Neuroscience and biobehavioral reviews
The past decade has produced an explosion in the number and variety of genetic tools available to neuroscientists, resulting in an unprecedented ability to precisely manipulate the genome and epigenome in behaving animals. However, no single resource exists that describes all of the tools available to neuroscientists. Here, we review the genetic, transgenic, and viral techniques that are currently available to probe the complex relationship between genes and cognition. Topics covered include types of traditional transgenic mouse models (knockout, knock-in, reporter lines), inducible systems (Cre-loxP, Tet-On, Tet-Off) and cell- and circuit-specific systems (TetTag, TRAP, DIO-DREADD). Additionally, we provide details on virus-mediated and siRNA/shRNA approaches, as well as a comprehensive discussion of the myriad manipulations that can be made using the CRISPR-Cas9 system, including single base pair editing and spatially- and temporally-regulated gene-specific transcriptional control. Collectively, this review will serve as a guide to assist neuroscientists in identifying and choosing the appropriate genetic tools available to study the complex relationship between the brain and behavior.
Applications of CRISPR Genome Engineering in Cell Biology.
Wang Fangyuan,Qi Lei S
Trends in cell biology
Recent advances in genome engineering are starting a revolution in biological research and translational applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease CRISPR associated protein 9 (Cas9) and its variants enable diverse manipulations of genome function. In this review, we describe the development of Cas9 tools for a variety of applications in cell biology research, including the study of functional genomics, the creation of transgenic animal models, and genomic imaging. Novel genome engineering methods offer a new avenue to understand the causality between the genome and phenotype, thus promising a fuller understanding of cell biology.
CRISPR/Cas9-mediated genome editing in nonhuman primates.
Kang Yu,Chu Chu,Wang Fang,Niu Yuyu
Disease models & mechanisms
Owing to their high similarity to humans, non-human primates (NHPs) provide an exceedingly suitable model for the study of human disease. In this Review, we summarize the history of transgenic NHP models and the progress of CRISPR/Cas9-mediated genome editing in NHPs, from the first proof-of-principle green fluorescent protein-expressing monkeys to sophisticated NHP models of human neurodegenerative disease that accurately phenocopy several complex disease features. We discuss not only the breakthroughs and advantages, but also the potential shortcomings of the application of the CRISPR/Cas9 system to NHPs that have emerged from the expanded understanding of this technology in recent years. Although off-target and mosaic mutations are the main concerns in CRISPR/Cas9-mediated NHP modeling, recent progress in genome editing techniques make it likely that these technical limitations will be overcome soon, bringing excellent prospects to human disease studies.
Editing the immune system in vivo in mice using CRISPR/Cas9 ribonucleoprotein (RNP)-mediated gene editing of transplanted hematopoietic stem cells.
Wang Rui,Graham Sean,Gao Lei,Tam Jason,Levesque Marc C
Methods (San Diego, Calif.)
CRISPR/Cas9-based genome editing has been widely used to evaluate target gene function in biomedical research. The CRISPR/Cas9 system can introduce gene knockout, knock-in and mutations with more ease than earlier generations of genome editing tools. Using CRISPR/Cas9-based genome editing, researchers have successfully modified the DNA of different immune components, including primary T cells, B cells, macrophages, and immune system progenitors, i.e. hematopoietic stem cells (HSCs), which are also known as Lin-Sca1+Kit+ cells (LSKs) in mice. We previously reported that the transplantation of HSCs with lentivirus-mediated CRISPR/Cas9-based genetic modifications into lethally irradiated congenic mice repopulated the ablated recipient immune system with the donor immune system. In this report, we efficiently manipulated CD40 expression in LSK cells using Cas9 RNP and demonstrated the functional impact in a colitis model. Compared to a virus-based strategy, the RNP approach has the potential to enable investigation of target gene biology in any mouse strain and eliminates the time and effort associated with virus production and infection. Therefore, in vivo RNP-based CRISPR/Cas9 gene editing of transplanted HSCs represents a promising new strategy for exploring gene function in the immune system of mice.
Progress on genome-wide CRISPR/Cas9 screening for functional genes and regulatory elements.
Liu Si Yuan,Yi Guo Qiang,Tang Zhong Lin,Chen Bin
Yi chuan = Hereditas
The CRISPR/Cas9 system is a powerful tool which has been extensively used for genome editing in the past few years. Nuclease-dead Cas9 (CRISPR/dCas9), a Cas9 protein mutant without splicing ability, along with loss-of- function (LOF), gain-of-function (GOF), or non-coding genes scanning approaches can reveal genome-scale functional determinants. CRISPR/Cas9 has been widely adopted to decipher disease mechanisms and pinpoint drug targets in the life science field, and also provide novel insights into animal genetics and breeding. In this review, we summarize the research progress in high-throughput CRISPR/Cas9 screening for revealing the functional genes and regulatory elements in the whole genome. We also highlight the applications of CRISPR/Cas9 system in the animal cells, providing a reference for gene editing and other related research in related fields.
CRISPR/Cas9 Technology as a Modern Genetic Manipulation Tool for Recapitulating of Neurodegenerative Disorders in Large Animal Models.
Barazesh Mahdi,Mohammadi Shiva,Bahrami Yadollah,Mokarram Pooneh,Morowvat Mohammad Hossein,Saidijam Massoud,Karimipour Morteza,Pour Soudabe Kavousi,Vosoughi Amir Reza,Khanaki Korosh
Current gene therapy
BACKGROUND:Neurodegenerative diseases are often the consequence of alterations in structures and functions of the Central Nervous System [CNS] in patients. Despite obtaining massive genomic information concerning the molecular basis of these diseases and since the neurological disorders are multifactorial, causal connections between pathological pathways at molecular level and CNS disorders development have remained obscure and need to be elucidated to a great extent. OBJECTIVE:Animal models serve as accessible and valuable tools for understanding and discovering the roles of causative factors in the development of neurodegenerative disorders and finding appropriate treatments. Contrary to rodents and other small animals, large animals especially non-human primates [NHPs] are remarkably alike humans; hence, they establish suitable models for recapitulating the main human's neuropathological manifestations that may not be seen in rodent models. Also, they serve as useful models to discover effective therapeutic targets for neurodegenerative disorders due to their similarity to humans in terms of physiology, evolutionary distance, anatomy, and behavior. METHOD:In this review, we recommend different strategies based on the CRISPR-Cas9 system for generating animal models of human neurodegenerative disorders and explain in vivo CRISPR-Cas9 delivery procedures of that are applied to disease models for therapeutic purposes. RESULTS:With the emergence of CRISPR/Cas9 as a modern specific gene-editing technology in the field of genetic engineering, genetic modification procedures such as gene knock-in and knock-out have become increasingly easier compared to traditional gene targeting techniques. Unlike the old techniques, this versatile technology can efficiently generate transgenic large animal models without need to complicate lab instruments. Hence, these animals can accurately replicate the signs of neurodegenerative disorders. CONCLUSION:Preclinical applications of CRISPR/Cas9 gene-editing technology supply a unique opportunity to establish animal models of neurodegenerative disorders with high accuracy and facilitate perspectives for breakthroughs in the research on the nervous system disease therapy and drug discovery. Furthermore, the useful outcomes of CRISPR applications in various clinical phases are hopeful for their translation to the clinic in a short time.
Zygote Electroporation for CRISPR/Cas9 Delivery to Generate Genetically Modified Mice.
Methods in molecular biology (Clifton, N.J.)
The CRISPR/Cas9 system is a powerful tool for generation of genetically modified mice. In conventional protocols, Cas9 protein (or mRNA) and sgRNA are introduced into zygotes by microinjection. However, microinjection requires special skill and is too time-consuming to treat zygotes on a large scale. Recently, we have developed a simple electroporation method which generates genetically modified mice with high efficiency. Here, we describe our method GEEP (genome editing by electroporation of Cas9 protein). This method facilitates high-throughput genetic analysis of the mouse. This chapter describes the GEEP method to generate genetically modified mice.