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: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:The prime editing (PE) system consists of a Cas9 nickase fused to a reverse transcriptase, which introduces precise edits into the target genomic region guided by a prime editing guide RNA. However, PE efficiency is limited by mismatch repair. To overcome this limitation, transient expression of a dominant-negative MLH1 (MLH1dn) has been used to inhibit key components of mismatch repair. Here, we designed a de novo MLH1 small binder (MLH1-SB) that binds to the dimeric interface of MLH1 and PMS2 using RFdiffusion and AlphaFold 3. The compact size of MLH1-SB enabled its integration into existing PE architectures via 2A systems, creating a novel PE-SB platform. The PE7-SB system significantly improved PE efficiency, achieving an 18.8-fold increase over PEmax and a 2.5-fold increase over PE7 in HeLa cells, as well as a 3.4-fold increase over PE7 in mice. This study highlights the potential of generative AI in advancing genome editing technology.
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Project description:Epigenetic correction of defective plasticity in a tauopathy mouse model with an acetyltransferase activator
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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:CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial immunity-related systems, each involving DNA modification. The Cas9 endonuclease introduces DNA double-strand breaks (DSBs), and the A. tumefaciens T-DNA is released by the VirD2 endonuclease assisted by VirDl and attached by VirE2, transferred to the plant nucleus and integrated into the genome. Here, we explored the potential for synergy between the two systems and found that Cas9 and three virulence (Vir) proteins achieve precise genome editing via the homology directed repair (HDR) pathway in tobacco and rice plants. Compared with Cas9T (Cas9, VirD1, VirE2) and CvD (Cas9-VirD2) systems, the HDR frequencies of a foreign GFPm gene in the CvDT system (Cas9-VirD2, VirD1, VirE2) increased 52-fold and 22-fold, respectively. Further optimization of the CvDT process with a donor linker (CvDTL) achieved a remarkable increase in the efficiency of HDR-mediated genome editing. Additionally, the HDR efficiency of the three rice endogenous genes ACETOLACTATE SYNTHASE (ALS), PHYTOENE DESATURASE (PDS), and NITROGEN TRANSPORTER 1.1 B (NRT1.1B) increased 24-, 32- and 16-fold, respectively, in the CvDTL system, compared with corresponding Cas9TL (Cas9T process with a donor linker). Our results suggest that collaboration between CRISPR/Cas9 and Agrobacterium-mediated genetic transformation can make great progress towards highly efficient and precise genome editing via the HDR pathway.
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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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