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Crispr screen

Xiaoping Su, Kuiqing Cui, Shanshan Du, Hongli Li, Fenghua Lu, Deshun Shi, Qingyou Liu
Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system...
March 19, 2018: In Vitro Cellular & Developmental Biology. Animal
Ferdinand Roesch, Molly OhAinle, Michael Emerman
The InterFeron Induced TransMembrane (IFITM) proteins are interferon stimulated genes that restrict many viruses, including HIV-1. SAMHD1 is another restriction factor blocking replication of HIV-1 and other viruses. Some lentiviruses evolved Vpx/Vpr proteins to degrade SAMHD1. However, this viral antagonism can be perturbed by host mechanisms: a recent study showed that in interferon (IFN) treated THP1 cells, Vpx is unable to degrade SAMHD1. In the present work, we designed an Interferon Stimulated Genes (ISGs)-targeted CRISPR knockout screen in order to identify ISGs regulating this phenotype...
March 20, 2018: Retrovirology
Vid Leko, Smitha Sripathy, Robin L Adrianse, Taylor Loe, Angela Park, Uyen Lao, Eric J Foss, Marisa S Bartolomei, Antonio Bedalov
Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi)...
March 2, 2018: Journal of Visualized Experiments: JoVE
John A Calarco, Adam D Norris
Genetic interaction screens are a powerful methodology to establish novel roles for genes and elucidate functional connections between genes. Such studies have been performed to great effect in single-cell organisms such as yeast and E. coli (Schuldiner et al ., 2005; Butland et al ., 2008; Costanzo et al ., 2010), but similar large-scale interaction studies using targeted reverse-genetic deletions in multi-cellular organisms have not been feasible. We developed a CRISPR/Cas9-based method for deleting genes in C...
March 5, 2018: Bio-protocol
Christopher J Lambert, Briana C Freshner, Arlen Chung, Tamara J Stevenson, D Miranda Bowles, Raheel Samuel, Bruce K Gale, Joshua L Bonkowsky
Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype...
2018: PloS One
Neftali Vazquez, Lilia Sanchez, Rebecca Marks, Eduardo Martinez, Victor Fanniel, Alma Lopez, Andrea Salinas, Itzel Flores, Jesse Hirschmann, Robert Gilkerson, Erin Schuenzel, Robert Dearth, Reginald Halaby, Wendy Innis-Whitehouse, Megan Keniry
BACKGROUND: Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone...
March 14, 2018: BMC Molecular Biology
Katia Tarasava, Rongming Liu, Andrew Garst, Ryan T Gill
Optimization of metabolic flux is a difficult and time-consuming process that often involves changing the expression levels of multiple genes simultaneously. While some pathways have a known rate limiting step, more complex metabolic networks can require a trial-and-error approach of tuning the expression of multiple genes to achieve a desired distribution of metabolic resources. Here we present an efficient method for generating expression diversity on a combinatorial scale using CRISPR interference. We use a modified native Escherichia coli Type I-E CRISPR-Cas system and an iterative cloning strategy for construction of guide RNA arrays...
March 14, 2018: Biotechnology and Bioengineering
Longzheng Chen, Wei Li, Lorenzo Katin-Grazzini, Jing Ding, Xianbin Gu, Yanjun Li, Tingting Gu, Ren Wang, Xinchun Lin, Ziniu Deng, Richard J McAvoy, Frederick G Gmitter, Zhanao Deng, Yunde Zhao, Yi Li
Developing CRISPR/Cas9-mediated non-transgenic mutants in asexually propagated perennial crop plants is challenging but highly desirable. Here, we report a highly useful method using an Agrobacterium -mediated transient CRISPR/Cas9 gene expression system to create non-transgenic mutant plants without the need for sexual segregation. We have also developed a rapid, cost-effective, and high-throughput mutant screening protocol based on Illumina sequencing followed by high-resolution melting (HRM) analysis. Using tetraploid tobacco as a model species and the phytoene desaturase ( PDS ) gene as a target, we successfully created and expediently identified mutant plants, which were verified as tetra-allelic mutants...
2018: Horticulture Research
Yusuke Tarumoto, Bin Lu, Tim D D Somerville, Yu-Han Huang, Joseph P Milazzo, Xiaoli S Wu, Olaf Klingbeil, Osama El Demerdash, Junwei Shi, Christopher R Vakoc
The lineage-specific transcription factor (TF) MEF2C is often deregulated in leukemia. However, strategies to target this TF have yet to be identified. Here, we used a domain-focused CRISPR screen to reveal an essential role for LKB1 and its Salt-Inducible Kinase effectors (SIK3, in a partially redundant manner with SIK2) to maintain MEF2C function in acute myeloid leukemia (AML). A key phosphorylation substrate of SIK3 in this context is HDAC4, a repressive cofactor of MEF2C. Consequently, targeting of LKB1 or SIK3 diminishes histone acetylation at MEF2C-bound enhancers and deprives leukemia cells of the output of this essential TF...
February 28, 2018: Molecular Cell
Tyler S Klann, Gregory E Crawford, Timothy E Reddy, Charles A Gersbach
Genomic regulatory elements that control gene expression play an important role in many traits and diseases. Identifying the regulatory elements associated with each gene or phenotype and understanding the function of that element remain a significant challenge. To address this technological need, we developed CRISPR/Cas9-based epigenomic regulatory element screening (CERES) for improved high-throughput screening of regulatory element activity in the native genomic context. This protocol includes detailed instructions for design and cloning of gRNA libraries, construction of endogenous reporter cell lines via CRISPR/Cas9-mediated knock-in of fluorescent proteins, overall screen design, and recovery of the gRNA library for enrichment analysis...
2018: Methods in Molecular Biology
Nicholas J Kramer, Michael S Haney, David W Morgens, Ana Jovičić, Julien Couthouis, Amy Li, James Ousey, Rosanna Ma, Gregor Bieri, C Kimberly Tsui, Yingxiao Shi, Nicholas T Hertz, Marc Tessier-Lavigne, Justin K Ichida, Michael C Bassik, Aaron D Gitler
Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons...
March 5, 2018: Nature Genetics
Guangchuan Wang, Ryan D Chow, Lupeng Ye, Christopher D Guzman, Xiaoyun Dai, Matthew B Dong, Feng Zhang, Phillip A Sharp, Randall J Platt, Sidi Chen
Cancer genomics consortia have charted the landscapes of numerous human cancers. Whereas some mutations were found in classical oncogenes and tumor suppressors, others have not yet been functionally studied in vivo. To date, a comprehensive assessment of how these genes influence oncogenesis is lacking. We performed direct high-throughput in vivo mapping of functional variants in an autochthonous mouse model of cancer. Using adeno-associated viruses (AAVs) carrying a single-guide RNA (sgRNA) library targeting putative tumor suppressor genes significantly mutated in human cancers, we directly pool-mutagenized the livers of Cre-inducible CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) mice...
February 2018: Science Advances
Tyler S Klann, Joshua B Black, Charles A Gersbach
Developments in CRISPR/Cas9-based technologies provide a new paradigm in functional screening of the genome. Conventional screening methods have focused on high-throughput perturbations of the protein-coding genome with technologies such as RNAi. However, equivalent methods for perturbing the non-coding genome have not existed until recently. CRISPR-based screening of genomic DNA has enabled the study of both genes and non-coding gene regulatory elements. Here we review recent progress in assigning function to the non-coding genome using CRISPR-based genomic and epigenomic screens, and discuss the prospects of these technologies to transforming our understanding of genome structure and regulation...
February 28, 2018: Current Opinion in Biotechnology
Yibo Zhang, Zhiwei Zhang, Wei Ge
Homology-directed recombination (HDR)-mediated genome editing is a powerful approach for both basic functional study and disease modeling. Although some studies have reported HDR-mediated precise editing in non-rodent models, the efficiency of establishing pure mutant animal lines that carry specific amino acid substitutions remains low. Furthermore, because the efficiency of nonhomologous end joining (NHEJ)-induced insertion and deletion (indel) mutations is normally much higher than that of HDR-induced point mutations, it is often difficult to identify the latter in the background of indel mutations...
March 2, 2018: Journal of Biological Chemistry
Ruimin Gao, Biruk A Feyissa, Mana Croft, Abdelali Hannoufa
The CRISPR/Cas9 technique was successfully used to edit the genome of the obligatory outcrossing plant species Medicago sativa L. (alfalfa). RNA-guided genome engineering using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 technology enables a variety of applications in plants. Successful application and validation of the CRISPR technique in a multiplex genome, such as that of M. sativa (alfalfa) will ultimately lead to major advances in the improvement of this crop. We used CRISPR/Cas9 technique to mutate squamosa promoter binding protein like 9 (SPL9) gene in alfalfa...
February 28, 2018: Planta
Bettina L Lee, Kathleen M Mirrashidi, Irma B Stowe, Sarah K Kummerfeld, Colin Watanabe, Benjamin Haley, Trinna L Cuellar, Michael Reichelt, Nobuhiko Kayagaki
The NLRC4 inflammasome recognizes bacterial flagellin and components of the type III secretion apparatus. NLRC4 stimulation leads to caspase-1 activation followed by a rapid lytic cell death known as pyroptosis. NLRC4 is linked to pathogen-free auto-inflammatory diseases, suggesting a role for NLRC4 in sterile inflammation. Here, we show that NLRC4 activates an alternative cell death program morphologically similar to apoptosis in caspase-1-deficient BMDMs. By performing an unbiased genome-wide CRISPR/Cas9 screen with subsequent validation studies in gene-targeted mice, we highlight a critical role for caspase-8 and ASC adaptor in an alternative apoptotic pathway downstream of NLRC4...
February 28, 2018: Scientific Reports
Florian Heigwer, Fillip Port, Michael Boutros
In the last decade, RNA interference (RNAi), a cellular mechanism that uses RNA-guided degradation of messenger RNA transcripts, has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans , RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila , RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale...
March 2018: Genetics
Xin-Yuan Qiu, Lv-Yun Zhu, Chu-Shu Zhu, Jia-Xin Ma, Tao Hou, Xiao-Min Wu, Si-Si Xie, Lu Min, De-An Tan, Dongyi Zhang, Lingyun Zhu
MicroRNAs have been reported related to multiple diseases and has potential applications in diagnosis and therapeutics. However, detection of miRNAs remains improvable, given their complexity, high cost and low sensitivity of currently. In this study, we attempt to build a novel platform that detects miRNAs at low cost and high efficacy. This detection system contains isothermal amplification, detecting and reporting process based on Rolling Circle Amplification, CRISPR-Cas9, and split-Horseradish Peroxidase techniques...
February 27, 2018: ACS Synthetic Biology
Takuji Yamauchi, Takeshi Masuda, Matthew C Canver, Michael Seiler, Yuichiro Semba, Mohammad Shboul, Mohammed Al-Raqad, Manami Maeda, Vivien A C Schoonenberg, Mitchel A Cole, Claudio Macias-Trevino, Yuichi Ishikawa, Qiuming Yao, Michitaka Nakano, Fumio Arai, Stuart H Orkin, Bruno Reversade, Silvia Buonamici, Luca Pinello, Koichi Akashi, Daniel E Bauer, Takahiro Maeda
To identify novel targets for acute myeloid leukemia (AML) therapy, we performed genome-wide CRISPR-Cas9 screening using AML cell lines, followed by a second screen in vivo. Here, we show that the mRNA decapping enzyme scavenger (DCPS) gene is essential for AML cell survival. The DCPS enzyme interacted with components of pre-mRNA metabolic pathways, including spliceosomes, as revealed by mass spectrometry. RG3039, a DCPS inhibitor originally developed to treat spinal muscular atrophy, exhibited anti-leukemic activity via inducing pre-mRNA mis-splicing...
February 8, 2018: Cancer Cell
Rongming Liu, Liya Liang, Andrew D Garst, Alaksh Choudhury, Violeta Sànchez I Nogué, Gregg T Beckham, Ryan T Gill
Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)H metabolism, and the aldehyde reduction system...
February 22, 2018: Metabolic Engineering
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