Increase genome editing efficiency by Cas9 transcriptional activator and histone acetyltransferase activator
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA827683 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18788900_1.fastq.gz | Fastqsanger.gz | |||
| SRR18788900_2.fastq.gz | Fastqsanger.gz | |||
| SRR18788901_1.fastq.gz | Fastqsanger.gz | |||
| SRR18788901_2.fastq.gz | Fastqsanger.gz | |||
| SRR18788902_1.fastq.gz | Fastqsanger.gz |
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Project description:The CRISPR/Cas9 system shows diverse levels of genome editing activities on eukaryotic genomic DNA targets, and experiments desire high-efficiency targets. Here we show that chromatin open status is a pivotal determinant of the Cas9 editing activity in mammalian cells, and increasing chromatin accessibility can efficiently improve Cas9 genome editing activity. However, the strategy that fusing the VP64 transcriptional activation domain at the C-terminus of Cas9 can only promote genome editing activity slightly at most tested CRISPR/Cas9 targets in Lenti-X 293T cells. Because histone acetylation increases eukaryotic chromatin accessibility, we further improve genome editing by elevating histone acetylation. We demonstrate that promoting histone acetylation using histone acetyltransferase (HAT) activator YF-2 can improve genome editing from Cas9 and more robustly from the Cas9 transcriptional activator. This provides a strategy to improve CRISPR/Cas9 genome editing activity and enables broader gRNA target choices in eukaryotes.
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Project description:Versatile and precise genome modifications are needed to create a wider range of adoptive cellular therapies1-5. Here we report two improvements that increase the efficiency of CRISPR-Cas9-based genome editing in clinically relevant primary cell types. Truncated Cas9 target sequences (tCTSs) added at the ends of the homology-directed repair (HDR) template interact with Cas9 ribonucleoproteins (RNPs) to shuttle the template to the nucleus, enhancing HDR efficiency approximately two- to fourfold. Furthermore, stabilizing Cas9 RNPs into nanoparticles with polyglutamic acid further improves editing efficiency by approximately twofold, reduces toxicity, and enables lyophilized storage without loss of activity. Combining the two improvements increases gene targeting efficiency even at reduced HDR template doses, yielding approximately two to six times as many viable edited cells across multiple genomic loci in diverse cell types, such as bulk (CD3+) T cells, CD8+ T cells, CD4+ T cells, regulatory T cells (Tregs), γδ T cells, B cells, natural killer cells, and primary and induced pluripotent stem cell-derived6 hematopoietic stem progenitor cells (HSPCs).
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Project description:Epigenetic modifications determine the structure and regulation of eukaryotic genomes and define key signatures of cell lineage specification. Technologies that facilitate the targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we have generated a programmable acetyltransferase based on the CRISPR/Cas9 gene regulation system, consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. This fusion protein catalyzes acetylation of histone H3 lysine 27 (H3K27) at its target sites, leading to robust transcriptional activation of target genes from promoters, proximal enhancers, and distal enhancers. In contrast to conventional dCas9-based activators, the acetyltransferase fusion effectively activated genes from enhancer regions and with individual guide RNAs. The core p300 domain was also portable to other programmable DNA-binding proteins. This technology enables the targeted perturbation of native epigenetic architecture and will be useful for reprogramming the epigenome for applications in genomics, genetics, disease modeling, and manipulating cell fate.
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Project description:MotivationThe development of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has provided a simple yet powerful system for targeted genome editing. In recent years, this system has been widely used for various gene editing applications. The CRISPR editing efficacy is mainly dependent on the single guide RNA (sgRNA), which guides Cas9 for genome cleavage. While there have been multiple attempts at improving sgRNA design, there is a pressing need for greater sgRNA potency and generalizability across various experimental conditions.ResultsWe employed a unique plasmid library expressed in human cells to quantify the potency of thousands of CRISPR/Cas9 sgRNAs. Differential sequence and structural features among the most and least potent sgRNAs were then used to train a machine learning algorithm for assay design. Comparative analysis indicates that our new algorithm outperforms existing CRISPR/Cas9 sgRNA design tools.Availability and implementationThe new sgRNA design tool is freely accessible as a web application, http://crispr.wustl.edu.Supplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Technologies that enable targeted manipulation of epigenetic marks could be used to precisely control cell phenotype or interrogate the relationship between the epigenome and transcriptional control. Here we describe a programmable, CRISPR-Cas9-based acetyltransferase consisting of the nuclease-null dCas9 protein fused to the catalytic core of the human acetyltransferase p300. The fusion protein catalyzes acetylation of histone H3 lysine 27 at its target sites, leading to robust transcriptional activation of target genes from promoters and both proximal and distal enhancers. Gene activation by the targeted acetyltransferase was highly specific across the genome. In contrast to previous dCas9-based activators, the acetyltransferase activates genes from enhancer regions and with an individual guide RNA. We also show that the core p300 domain can be fused to other programmable DNA-binding proteins. These results support targeted acetylation as a causal mechanism of transactivation and provide a robust tool for manipulating gene regulation.
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Project description:Efforts to improve CRISPR-Cas9 genome editing systems for lower off-target effects are mostly at the cost of its robust on-target efficiency. To enhance both accuracy and efficiency, we created chimeric SpyCas9 proteins fused with the 5'-to-3' exonuclease Recombination J (RecJ) or with GFP and demonstrated that transfection of the pre-assembled ribonucleoprotein of the two chimeric proteins into human or plant cells resulted in greater targeted mutagenesis efficiency up to 600% without noticeable increase in off-target effects. Improved activity of the two fusion proteins should enable editing of the previously hard-to-edit genes and thus readily obtaining the cells with designer traits.
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