The Experts below are selected from a list of 21189 Experts worldwide ranked by ideXlab platform
Michael Boutros - One of the best experts on this subject based on the ideXlab platform.
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crispr library designer cld software for multispecies design of single guide RNA Libraries
Genome Biology, 2016Co-Authors: Florian Heigwer, Tianzuo Zhan, Marco Breinig, Jan Winter, Dirk Brugemann, S Leible, Michael BoutrosAbstract:Background Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes.
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crispr library designer cld software for multispecies design of single guide RNA Libraries
Genome Biology, 2016Co-Authors: Florian Heigwer, Tianzuo Zhan, Marco Breinig, Jan Winter, Dirk Brugemann, S Leible, Michael BoutrosAbstract:Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes. Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) Libraries for all organisms with annotated genomes. CLD is suitable for the design of Libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs. CLD predicts a high fraction of functional sgRNAs and is publicly available at https://github.com/boutroslab/cld .
Tianzuo Zhan - One of the best experts on this subject based on the ideXlab platform.
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crispr library designer cld software for multispecies design of single guide RNA Libraries
Genome Biology, 2016Co-Authors: Florian Heigwer, Tianzuo Zhan, Marco Breinig, Jan Winter, Dirk Brugemann, S Leible, Michael BoutrosAbstract:Background Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes.
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crispr library designer cld software for multispecies design of single guide RNA Libraries
Genome Biology, 2016Co-Authors: Florian Heigwer, Tianzuo Zhan, Marco Breinig, Jan Winter, Dirk Brugemann, S Leible, Michael BoutrosAbstract:Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes. Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) Libraries for all organisms with annotated genomes. CLD is suitable for the design of Libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs. CLD predicts a high fraction of functional sgRNAs and is publicly available at https://github.com/boutroslab/cld .
Florian Heigwer - One of the best experts on this subject based on the ideXlab platform.
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crispr library designer cld software for multispecies design of single guide RNA Libraries
Genome Biology, 2016Co-Authors: Florian Heigwer, Tianzuo Zhan, Marco Breinig, Jan Winter, Dirk Brugemann, S Leible, Michael BoutrosAbstract:Background Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes.
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crispr library designer cld software for multispecies design of single guide RNA Libraries
Genome Biology, 2016Co-Authors: Florian Heigwer, Tianzuo Zhan, Marco Breinig, Jan Winter, Dirk Brugemann, S Leible, Michael BoutrosAbstract:Genetic screens using CRISPR/Cas9 are a powerful method for the functional analysis of genomes. Here we describe CRISPR library designer (CLD), an integrated bioinformatics application for the design of custom single guide RNA (sgRNA) Libraries for all organisms with annotated genomes. CLD is suitable for the design of Libraries using modified CRISPR enzymes and targeting non-coding regions. To demonstrate its utility, we perform a pooled screen for modulators of the TNF-related apoptosis inducing ligand (TRAIL) pathway using a custom library of 12,471 sgRNAs. CLD predicts a high fraction of functional sgRNAs and is publicly available at https://github.com/boutroslab/cld .
Jianping Chen - One of the best experts on this subject based on the ideXlab platform.
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genome wide characterization of rice black streaked dwarf virus responsive microRNAs in rice leaves and roots by small RNA and degradome sequencing
Plant and Cell Physiology, 2015Co-Authors: Zongtao Sun, Xu Wang, Jianping ChenAbstract:MicroRNAs (miRNAs) are small, non-coding RNAs which typically function by guiding cleavage of target mRNAs. They play important roles in development, abiotic stress and responses to pathogens. Four small RNA Libraries and four degradome Libraries were constructed from the leaves and roots of healthy rice and plants infected with Rice black streaked dwarf virus (RBSDV). Analysis of the deep sequencing results showed that the expression patterns of 14 miRNAs in leaves and 16 miRNAs in roots changed significantly in response to RBSDV infection. Some responses were similar in roots and leaves, but many miRNAs responded differently in different tissues. The results were confirmed for selected miRNAs by quantitative real-time PCR. By using degradome sequencing, a total of 104 target transcripts for 17 conserved and 16 non-conserved miRNAs were shown to be responsive to RBSDV infection. Fifteen novel miRNAs were also identified by small RNA and degradome sequencing. The results provide new insights into the regulatory networks of miRNAs and their targets in different plant tissues in response to virus infection.
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identification of novel oryza sativa miRNAs in deep sequencing based small RNA Libraries of rice infected with rice stripe virus
PLOS ONE, 2012Co-Authors: Weixia Guo, Fei Yan, Hongying Zheng, Lin Lin, Hairu Chen, Jianping ChenAbstract:MicroRNAs (miRNAs) play essential regulatory roles in the development of eukaryotes. Methods based on deep-sequencing have provided a powerful high-throughput strategy for identifying novel miRNAs and have previously been used to identify over 100 novel miRNAs from rice. Most of these reports are related to studies of rice development, tissue differentiation, or abiotic stress, but novel rice miRNAs related to viral infection have rarely been identified. In previous work, we constructed and pyrosequenced the small RNA (sRNA) Libraries of rice infected with Rice stripe virus and described the character of the small interfering RNAs (siRNA) derived from the RSV RNA genome. We now report the identification of novel miRNAs from the abundant sRNAs (with a minimum of 100 sequencing reads) in the sRNA library of RSV-infected rice. 7 putative novel miRNAs (pn-miRNAs) whose precursor sequences have not previously been described were identified and could be detected by Northern blot or RT-PCR, and were recognized as novel miRNAs (n-miRNAs). Further analysis showed that 5 of the 7 n-miRNAs were up-expressed while the other 2 n-miRNAs were down-expressed in RSV-infected rice. In addition, 23 pn-miRNAs that were newly produced from 19 known miRNA precursors were also identified. This is first report of novel rice miRNAs produced from new precursors related to RSV infection.
Axel Schambach - One of the best experts on this subject based on the ideXlab platform.
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pooled generation of lentiviral tetracycline regulated microRNA embedded short hairpin RNA Libraries
Human Gene Therapy Methods, 2018Co-Authors: Felix F Adams, Thomas Hoffmann, Johannes Zuber, Dirk Heckl, Axel Schambach, Adrian SchwarzerAbstract:Short hairpin RNA (shRNA) screens are powerful tools to probe genetic dependencies in loss-of-function studies, such as the identification of therapeutic targets in cancer research. Lentivirally delivered shRNAs embedded in endogenous microRNA contexts (shRNAmiRs) mediate efficient long-term suppression of target genes suitable for numerous experimental contexts and clinical applications. Here, an easy-to-use laboratory protocol is described, covering the design and pooled assembly of focused shRNAmiR Libraries into an optimized, Tet-inducible all-in-one lentiviral vector, packaging of viral particles, followed by retrieval and quantification of hairpin sequences after cellular DNA-recovery. Starting from a gene list to the identification of hits, the protocol enables shRNA screens within 6 weeks.
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an optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA Libraries
Biomaterials, 2017Co-Authors: Felix F Adams, Thomas Hoffmann, Johannes Zuber, Dirk Heckl, Axel Schambach, Steven R Talbot, Arnold Kloos, Felicitas Thol, Michael HeuserAbstract:Abstract RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR Libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system.
