Disruption of miRNA Sequence by TALENs and CRISPR/Cas9 Induces Varied Length of miRNA Production [TALENs]
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA534426 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8948940.fastq.gz | Fastqsanger.gz | |||
| SRR8948941.fastq.gz | Fastqsanger.gz | |||
| SRR8948942.fastq.gz | Fastqsanger.gz | |||
| SRR8948943.fastq.gz | Fastqsanger.gz | |||
| SRR8948936.fastq.gz | Fastqsanger.gz |
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Project description:Disruption of miRNA Sequence by TALENs and CRISPR/Cas9 Induces Varied Length of miRNA Production [CRISPR/Cas9]
| PRJNA540493 | ENA
Project description:Purpose: MicroRNAs (miRNAs) have been recognized as crucial players in plant growth, development, production and responses to biotic and abiotic stresses, while only limited number of mutants can be used to dissect their roles. Here we used TALENs (Transcription Activator-Like Effector Nucleases) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce base pair mutations in MIRNA genes, resulting in creation of miRNA mutants and elucidating the effect of mutations on the miRNA biogenesis in rice and Arabidopsis. Methods: Total RNAs were prepared using a Trizol reagent-based method. The plant materials were homogenized and reconstituted in Trizol solution. The solution in turn was extracted by chloroform. Isopropanol was used to precipitate the total RNA. The whole process was performed under RNase free environment. DEPC-treated water was used to dissolve RNA. The total RNA underwent miRNA isolation using Qiagen miRNA purification kit according to the manufacturer’s manual. Small RNA libraries were made using 5 µg total RNA with Illumina TruSeq Small RNA Library Prep Kit as described, and sequenced on the Illumina HiSeq 2500 platform.
2019-05-01 | GSE130510 | GEO
Project description:Purpose: MicroRNAs (miRNAs) have been recognized as crucial players in plant growth, development, production and responses to biotic and abiotic stresses, while only limited number of mutants can be used to dissect their roles. Here we used TALENs (Transcription Activator-Like Effector Nucleases) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce base pair mutations in MIRNA genes, resulting in creation of miRNA mutants and elucidating the effect of mutations on the miRNA biogenesis in rice and Arabidopsis. Methods: Total RNAs were prepared using a Trizol reagent-based method. The plant materials were homogenized and reconstituted in Trizol solution. The solution in turn was extracted by chloroform. Isopropanol was used to precipitate the total RNA. The whole process was performed under RNase free environment. DEPC-treated water was used to dissolve RNA. The total RNA underwent miRNA isolation using Qiagen miRNA purification kit according to the manufacturer’s manual. Small RNA libraries were made using 5 µg total RNA with NEBNext Small RNA library Prep Set, as described, and sequenced on the Illumina HiSeq 2500 platform.
2019-04-25 | GSE130239 | GEO
Project description:MicroRNAs (miRNAs) are 20-24 nucleotides (nt) small RNAs functioning in eukaryotes. The length and sequence of miRNAs are not only related to the biogenesis of miRNAs but are also important for downstream physiological processes like ta-siRNA production. To investigate these roles, it is informative to create small mutations within mature miRNA sequences. We used both TALENs (transcription activator-like effector nucleases) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) to introduce heritable base pair mutations in mature miRNA sequences. For rice, TALEN constructs were built targeting five different mature miRNA sequences and yielding heritable mutations. Among the resulting mutants, mir390 mutant showed a severe defect in the shoot apical meristem (SAM), a shootless phenotype, which could be rescued by the wild-type MIR390. Small RNA sequencing showed the two base pair deletion in mir390 substantially interfered with miR390 biogenesis. In Arabidopsis, CRISPR/Cas9-mediated editing of the miR160* strand confirmed that the asymmetric structure of miRNA is not a necessary determinant for secondary siRNA production. CRISPR/Cas9 with double-guide RNAs successfully generated mir160a null mutants with fragment deletions, at a higher efficiency than a single-guide RNA. The difference between the phenotypic severity of miR160a mutants in Col-0 versus Ler backgrounds highlights a diverged role for miR160a in different ecotypes. Overall, we demonstrated that TALENs and CRISPR/Cas9 are both effective in modifying miRNA precursor structure, disrupting miRNA processing and generating miRNA null mutant plants.
| S-EPMC7292542 | biostudies-literature
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. A subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy
2015-05-07 | GSE68620 | GEO
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. A subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy Four Huh7 cells lines at 3 different passages were analyzed. The reference cell line was Huh7 wild type cells (WT). The other three cell lines had an integration of an anti-HCV shmiRNA in the hcr locus and miR-122 intact (T2 31.3) or mutated (TS 30.20 and U6 20.16). RNA was extracted from three different passages.
2015-05-07 | E-GEOD-68620 | biostudies-arrayexpress
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. A subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy
2015-05-07 | GSE68637 | GEO
Project description:MicroRNA induces deadenylation of its targets according to the current model in the scientific community, but this model is based on the studies of a few individual genes. We tested the model by examining the global effect of miRNA on poly(A) tail of the targets. Synthetic miR-1 mimic was transfected into HeLa cells, and the poly(A) length was measured by TAIL-seq. Deadenylation of miR-1 targets was evident as early as 3 hours post-transfection without a significant change in mRNA level. After 6 or 9 hours, target mRNA level was substantially downregulated. Therefore, although there are some exceptions, our result confirms that, in general, miRNA indeed induces deadenylation. Furthermore, our kinetic global analysis confirms that deadenylation precedes mRNA decay.
2014-02-28 | GSE54114 | GEO
Project description:MicroRNA induces deadenylation of its targets according to the current model in the scientific community, but this model is based on the studies of a few individual genes. We tested the model by examining the global effect of miRNA on poly(A) tail of the targets. Synthetic miR-1 mimic was transfected into HeLa cells, and the poly(A) length was measured by TAIL-seq. Deadenylation of miR-1 targets was evident as early as 3 hours post-transfection without a significant change in mRNA level. After 6 or 9 hours, target mRNA level was substantially downregulated. Therefore, although there are some exceptions, our result confirms that, in general, miRNA indeed induces deadenylation. Furthermore, our kinetic global analysis confirms that deadenylation precedes mRNA decay. Four culture dishes of HeLa cells were treated with miR-1 all together, then collected and prepared for sequencing after different time of incubation (0, 3, 6, and 9 hours post-transfection)
2014-02-28 | E-GEOD-54114 | biostudies-arrayexpress
Project description:Short hairpin RNA (shRNA) expression strategies that allow safe and persistent target mRNA knockdown are key to the success of many in vitro or in vivo RNAi applications. Here, we propose a novel solution which is expression of a promoterless miRNA-adapted shRNA (shmiRNA) from an engineered genomic miRNA locus. For proof-of-concept, we genetically “vaccinated” liver cells against a human pathogen, by using TALEns or CRISPR to integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr gene. Reporter assays and qRT-PCR confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality. Specificity and safety of shmiRNA integration were validated via PCR, cDNA and miRNA profiling, and whole genome sequencing. A subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNA expression technologies should benefit numerous applications, from basic miRNA research, to human cell and gene therapy Four Huh7 cells lines at 3 different passages were analyzed. The reference cell line was Huh7 wild type cells (WT). The other three cell lines had an integration of an anti-HCV shmiRNA in the hcr locus and miR-122 intact (T2 31.3) or mutated (TS 30.20 and U6 20.16). RNA was extracted from three different passages.
2015-05-07 | E-GEOD-68637 | biostudies-arrayexpress