Ontology highlight
ABSTRACT:
PROVIDER: PRJNA489777 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7801930_1.fastq.gz | Fastqsanger.gz | |||
| SRR7801930_2.fastq.gz | Fastqsanger.gz | |||
| SRR7801931_1.fastq.gz | Fastqsanger.gz | |||
| SRR7801931_2.fastq.gz | Fastqsanger.gz | |||
| SRR7801932_1.fastq.gz | Fastqsanger.gz |
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Project description:Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically-relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identified putative immunogenic epitopes on each nuclease. Then, we used computational modeling to rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants were substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, while maintaining wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 was comparable in efficiency to wild-type SaCas9, but significantly reduced undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection.
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Project description:We assessed genome editing strategies to treat cardiomyopathy in mice including one or two doses of ABE8e, or one low, medium, or high dose of SaCas9 nuclease We conducted RNA analysis and genomic DNA analysis at the target region and transcriptome-wide analysis to assess cellular phenotypes
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Project description:Adenoid B cells from anonymous human donors were isolated and nucleofected with RNP complexes targeting two loci within exon 2 of the CD46 gene, which encodes a cell surface protein. One hour after nucleofection, the same number of control cells (w/o-RNP) and CD46-RNP complex treated cells were infected with Epstein-Barr virus (strain 6008_B95-8) with a multiplicity of infection of 0.1. Newly transcribed RNAs were metabolically labeled with 4-thiouridine (4sU) for 60 min 23 hours after EBV infection. Twenty-four hours after the start of the experiment, 4sU-biotin-labeled, newly transcribed RNAs were thiol-specifically biotinylated and separated from unlabeled RNA using Streptavidin beads. RNA-seq experiments with the enriched RNA samples were performed to analyze the edited CD46 locus encompassing the RNP complex target sites. The experiment revealed that 24 hours after CRISPR-Cas9 mediated gene editing a substantial fraction of CD46 transcript contained indels and a larger deletion in between the two targets sites within exon 2 of CD46.
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Project description:Optimization of CRISPR/Cas9-mediated genome engineering has resulted in base editors that hold promise for mutation repair and disease modeling. Here, we demonstrate the application of base editors for the generation of complex tumor models in human ASC-derived organoids. First we show Efficacy of cytosine and adenine base editors in modelingCTNNB1hot-spot mutations in hepatocyte organoids. Next, we use C>T base editors to insert nonsense mutations inPTENin endometrial organoids and demonstrate tumorigenicity even in the heterozygous state. Moreover, drug screening assays on organoids harboring eitherPTENorPTENandPIK3CAmutations reveal the mechanism underlying the initial stages of endometrial tumorigenesis. To further increase the scope of base editing we combine SpCas9 and SaCas9 for simultaneous C>T and A>G editing at individual target sites. Finally, we show that base editor multiplexing allow modeling of colorectal tumorigenesis in a single step by simultaneously transfecting sgRNAs targeting five cancer genes.
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