RNA-guided retargeting of Sleeping Beauty transposition in human cells

ABSTRACTTwo different approaches of genomic modification are currently used for genome engineering and gene therapy: integrating vectors, which can efficiently integrate large transgenes but are unspecific with respect to their integration sites, and nuclease-based approaches, which are highly specific but not efficient at integrating large genetic cargoes. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection of SB by fusing it to a catalytically inactive Cas9 (dCas9) and by providing a single guide RNA (sgRNA) against the human Alu retrotransposon. Enrichment of transposon integrations was dependent on the sgRNA, occurred in a relatively narrow, ∼200 bp window around the targeted sites and displayed an asymmetric pattern with a bias towards sites that are downstream of the sgRNA targets. Our data indicate that the targeting mechanism specified by CRISPR/Cas9 forces integration into genomic regions that are otherwise poor targets for SB transposition. Future modifications of this technology may allow the development of methods for efficient and specific gene insertion for precision genetic engineering.

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Genome-Wide Association Study of Body Weight Traits in Chinese Fine-Wool Sheep

Animals ◽

10.3390/ani10010170 ◽

2020 ◽

Vol 10 (1) ◽

pp. 170 ◽

Cited By ~ 2

Author(s):

Zengkui Lu ◽

Yaojing Yue ◽

Chao Yuan ◽

Jianbin Liu ◽

Zhiqiang Chen ◽

...

Keyword(s):

Body Weight ◽

Muscle Development ◽

Genome Wide Association Study ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Genome Wide ◽

Economic Trait ◽

Significance Levels ◽

Genomic Regions ◽

Selection Of

Body weight is an important economic trait for sheep and it is vital for their successful production and breeding. Therefore, identifying the genomic regions and biological pathways that contribute to understanding variability in body weight traits is significant for selection purposes. In this study, the genome-wide associations of birth, weaning, yearling, and adult weights of 460 fine-wool sheep were determined using resequencing technology. The results showed that 113 single nucleotide polymorphisms (SNPs) reached the genome-wide significance levels for the four body weight traits and 30 genes were annotated effectively, including AADACL3, VGF, NPC1, and SERPINA12. The genes annotated by these SNPs significantly enriched 78 gene ontology terms and 25 signaling pathways, and were found to mainly participate in skeletal muscle development and lipid metabolism. These genes can be used as candidate genes for body weight in sheep, and provide useful information for the production and genomic selection of Chinese fine-wool sheep.

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MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9 for enChIP analysis

10.21203/rs.2.16753/v4 ◽

2021 ◽

Author(s):

Miyuki Yuno ◽

Toshitsugu Fujita ◽

Hodaka Fujii

Keyword(s):

Dna Binding ◽

Streptococcus Pyogenes ◽

Chromatin Immunoprecipitation ◽

Genomic Region ◽

Target Cells ◽

Drug Selection ◽

Guide Rna ◽

Inactive Form ◽

Genomic Regions ◽

Selection Of

Abstract Engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, e.g., a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that by using these plasmids, it is feasible to purify target genomic regions with yields comparable to those reported using other systems. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells.

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Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA

PLANT PHYSIOLOGY ◽

10.1104/pp.15.00793 ◽

2015 ◽

Vol 169 (2) ◽

pp. 931-945 ◽

Cited By ~ 319

Author(s):

Sergei Svitashev ◽

Joshua K. Young ◽

Christine Schwartz ◽

Huirong Gao ◽

S. Carl Falco ◽

...

Keyword(s):

Gene Editing ◽

Targeted Mutagenesis ◽

Specific Gene ◽

Site Specific ◽

Guide Rna ◽

Gene Insertion

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SETBP1 overexpression acts in the place of class-defining mutations to drive FLT3-ITD–mutant AML

Blood Advances ◽

10.1182/bloodadvances.2020003443 ◽

2021 ◽

Vol 5 (9) ◽

pp. 2412-2425

Author(s):

Suruchi Pacharne ◽

Oliver M. Dovey ◽

Jonathan L. Cooper ◽

Muxin Gu ◽

Mathias J. Friedrich ◽

...

Keyword(s):

Insertional Mutagenesis ◽

Cancer Genomics ◽

Sleeping Beauty ◽

Sequencing Data ◽

Therapeutic Approaches ◽

Guide Rna ◽

Genome Wide ◽

Rna Targeting ◽

Transposon Insertions ◽

Tandem Duplications

Abstract Advances in cancer genomics have revealed genomic classes of acute myeloid leukemia (AML) characterized by class-defining mutations, such as chimeric fusion genes or in genes such as NPM1, MLL, and CEBPA. These class-defining mutations frequently synergize with internal tandem duplications in FLT3 (FLT3-ITDs) to drive leukemogenesis. However, ∼20% of FLT3-ITD–positive AMLs bare no class-defining mutations, and mechanisms of leukemic transformation in these cases are unknown. To identify pathways that drive FLT3-ITD mutant AML in the absence of class-defining mutations, we performed an insertional mutagenesis (IM) screening in Flt3-ITD mice, using Sleeping Beauty transposons. All mice developed acute leukemia (predominantly AML) after a median of 73 days. Analysis of transposon insertions in 38 samples from Flt3-ITD/IM leukemic mice identified recurrent integrations at 22 loci, including Setbp1 (20/38), Ets1 (11/38), Ash1l (8/38), Notch1 (8/38), Erg (7/38), and Runx1 (5/38). Insertions at Setbp1 led exclusively to AML and activated a transcriptional program similar, but not identical, to those of NPM1-mutant and MLL-rearranged AMLs. Guide RNA targeting of Setbp1 was highly detrimental to Flt3ITD/+/Setbp1IM+, but not to Flt3ITD/+/Npm1cA/+, AMLs. Also, analysis of RNA-sequencing data from hundreds of human AMLs revealed that SETBP1 expression is significantly higher in FLT3-ITD AMLs lacking class-defining mutations. These findings propose that SETBP1 overexpression collaborates with FLT3-ITD to drive a subtype of human AML. To identify genetic vulnerabilities of these AMLs, we performed genome-wide CRISPR-Cas9 screening in Flt3ITD/+/Setbp1IM+ AMLs and identified potential therapeutic targets, including Kdm1a, Brd3, Ezh2, and Hmgcr. Our study gives new insights into epigenetic pathways that can drive AMLs lacking class-defining mutations and proposes therapeutic approaches against such cases.

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enChIP systems with MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9

10.21203/rs.2.16753/v2 ◽

2020 ◽

Author(s):

Miyuki Yuno ◽

Toshitsugu Fujita ◽

Hodaka Fujii

Keyword(s):

Dna Binding ◽

Chromatin Immunoprecipitation ◽

Genomic Region ◽

Target Cells ◽

Guide Rna ◽

Inactive Form ◽

Different Types ◽

High Yields ◽

Genomic Regions ◽

Selection Of

Abstract Objective: Engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, e.g., a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. Results: We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that it is feasible to purify target genomic regions with high yields using these plasmids. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells. In addition, they could be used to analyze different types of target cells.

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The MLL3/4 complexes and MiDAC act antagonistically as genome-wide regulators of H4K20ac to control a specific gene expression program

10.1101/2021.12.17.473155 ◽

2021 ◽

Author(s):

Xiaokang Wang ◽

Wojciech Rosikiewicz ◽

Yurii Sedkov ◽

Baisakhi Mondal ◽

Satish Kallappagoudar ◽

...

Keyword(s):

Gene Expression ◽

Physiological Role ◽

Specific Gene ◽

Physical Interaction ◽

Specific Gene Expression ◽

Genome Wide ◽

Histone H3k27 ◽

Genomic Regions ◽

Transcriptional Output ◽

Expression Program

The mitotic deacetylase complex MiDAC has recently been shown to play a vital physiological role in embryonic development and neurite outgrowth. However, how MiDAC functionally intersects with other chromatin-modifying regulators is poorly understood. Here, we describe a physical interaction between the histone H3K27 demethylase UTX, a complex-specific subunit of the enhancer-associated MLL3/4 complexes, and MiDAC. We demonstrate that UTX bridges the association of the MLL3/4 complexes and MiDAC by interacting with ELMSAN1, a scaffolding subunit of MiDAC. Our data shows that MiDAC constitutes a negative genome-wide regulator of H4K20ac, an activity which is counteracted by the MLL3/4 complexes. MiDAC and the MLL3/4 complexes co-localize at many genomic regions, that are enriched for H4K20ac and the enhancer marks H3K4me1, H3K4me2 and H3K27ac. We find that MiDAC antagonizes the recruitment of the MLL3/4 complexes to negatively regulate H4K20ac, H3K4me2 and H3K27ac resulting in transcriptional attenuation of associated genes. In summary, our findings provide a paradigm how the opposing roles of chromatin-modifying components, such as MiDAC and the MLL3/4 complexes, balance the transcriptional output of specific gene expression programs.

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enChIP systems with MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9

10.21203/rs.2.16753/v3 ◽

2020 ◽

Author(s):

Miyuki Yuno ◽

Toshitsugu Fujita ◽

Hodaka Fujii

Keyword(s):

Dna Binding ◽

Chromatin Immunoprecipitation ◽

Genomic Region ◽

Target Cells ◽

Guide Rna ◽

Inactive Form ◽

Different Types ◽

High Yields ◽

Genomic Regions ◽

Selection Of

Abstract Engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, e.g., a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that it is feasible to purify target genomic regions with high yields using these plasmids. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells. In addition, they could be used to analyze different types of target cells.

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enChIP systems with MSCV-based retroviral plasmids expressing 3xFLAG-Sp-dCas9

10.21203/rs.2.16753/v1 ◽

2019 ◽

Author(s):

Miyuki Yuno ◽

Toshitsugu Fujita ◽

Hodaka Fujii

Keyword(s):

Dna Binding ◽

Chromatin Immunoprecipitation ◽

Genomic Region ◽

Target Cells ◽

Guide Rna ◽

Inactive Form ◽

Different Types ◽

High Yields ◽

Genomic Regions ◽

Selection Of

Abstract Objective: Engineered DNA-binding molecule–mediated chromatin immunoprecipitation (enChIP) is a technology for purifying specific genomic regions to facilitate identification of their associated molecules, including proteins, RNAs, and other genomic regions. In enChIP, the target genomic region is tagged with engineered DNA-binding molecules, e.g., a variant of the clustered regularly interspaced short palindromic repeats (CRISPR) system consisting of a guide RNA (gRNA) and a catalytically inactive form of Cas9 (dCas9). In this study, to increase the flexibility of enChIP and expand the range of target cells, we generated murine stem cell virus (MSCV)-based retroviral plasmids for expressing dCas9. Results: We constructed MSCV-based retroviral plasmids expressing Streptococcus pyogenes dCas9 fused to a 3xFLAG-tag (3xFLAG-Sp-dCas9) and various drug resistance genes. We showed that it is feasible to purify target genomic regions with high yields using these plasmids. These systems might give enChIP users greater flexibility in choosing optimal systems for drug selection of transduced cells. In addition, they could be used to analyze different types of target cells.

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Multi-species, multi-transcription factor binding highlights conserved control of tissue-specific biological pathways

eLife ◽

10.7554/elife.02626 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 56

Author(s):

Benoit Ballester ◽

Alejandra Medina-Rivera ◽

Dominic Schmidt ◽

Mar Gonzàlez-Porta ◽

Matthew Carlucci ◽

...

Keyword(s):

Transcription Factor ◽

Association Studies ◽

Specific Gene ◽

Genome Wide Association Studies ◽

Tissue Specific ◽

Genome Wide ◽

Multiple Species ◽

Lipid Metabolism Genes ◽

Genomic Regions ◽

Regulatory Dna

As exome sequencing gives way to genome sequencing, the need to interpret the function of regulatory DNA becomes increasingly important. To test whether evolutionary conservation of cis-regulatory modules (CRMs) gives insight into human gene regulation, we determined transcription factor (TF) binding locations of four liver-essential TFs in liver tissue from human, macaque, mouse, rat, and dog. Approximately, two thirds of the TF-bound regions fell into CRMs. Less than half of the human CRMs were found as a CRM in the orthologous region of a second species. Shared CRMs were associated with liver pathways and disease loci identified by genome-wide association studies. Recurrent rare human disease causing mutations at the promoters of several blood coagulation and lipid metabolism genes were also identified within CRMs shared in multiple species. This suggests that multi-species analyses of experimentally determined combinatorial TF binding will help identify genomic regions critical for tissue-specific gene control.

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