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Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways

ABSTRACT: Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways

PROVIDER: PRJNA1042627 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR26872846_1.fastq.gz	Fastqsanger.gz
	SRR26872846_2.fastq.gz	Fastqsanger.gz
	SRR26872847_1.fastq.gz	Fastqsanger.gz
	SRR26872847_2.fastq.gz	Fastqsanger.gz
	SRR26872848_1.fastq.gz	Fastqsanger.gz

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Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [WES]

Project description:Precise genome editing is crucial for establishing isogenic human disease models and ex vivo stem cell therapy from the patient-derived human pluripotent stem cells (hPSCs). Unlike Cas9-mediated knock-in, cytosine base editor (CBE) and prime editor (PE) achieve the desirable gene correction without inducing DNA double strand breaks. However, hPSCs possess highly active DNA repair pathways and are particularly susceptible to p53-dependent cell death. These unique characteristics impede the efficiency of gene editing in hPSCs. Here, we demonstrate that dual inhibition of p53-mediated cell death and distinct activation of the DNA damage repair system upon DNA damage by CBE or PE additively enhanced editing efficiency in hPSCs. The BE4stem system comprised of dominant negative p53 (p53DD) and three UNG inhibitor (UGI), engineered to specifically diminish base excision repair (BER), improved CBE efficiency in hPSCs. Addition of dominant negative MLH1 to inhibit mismatch repair activity and p53DD in the conventional PE system also significantly enhanced PE efficiency in hPSCs. Thus, combined inhibition of the unique cellular cascades engaged in hPSCs upon gene editing could significantly enhance precise genome editing in these cells.

2025-01-01 | GSE248190 | GEO

Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [RNA-seq]

2024-04-22 | GSE247589 | GEO

Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [WES]

Project description:Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [WES]

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Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [RNA-seq]

Project description:Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways [RNA-seq]

| PRJNA1039785 | ENA

Enhancing Precise Genome Editing in Human Pluripotent Stem Cells through Dual Inhibition of DNA Damage Response and Repair Pathways

Project description:This SuperSeries is composed of the SubSeries listed below.

2024-04-22 | GSE248191 | GEO

Precise Genome Editing in vivo with Single, Canonical AAV Vectors. Encoding Guide RNA, Cas9, and Homologous Repair Donor

Project description:Adeno-associated virus (AAV) vectors are important delivery platforms for therapeutic genome editing but are severely constrained by cargo limits, especially for large effectors like Cas9s. Simultaneous delivery of multiple vectors can limit dose and efficacy and increase safety risks. The use of compact effectors has enabled single-AAV delivery of Cas9s with 1-3 guides for edits that use end-joining repair pathways, but many precise edits that correct disease-causing mutations in vivo require homology-directed repair (HDR) templates. Here, we describe single-vector, ~4.8-kb AAV platforms that express Nme2Cas9 and either two sgRNAs to produce segmental deletions, or a single sgRNA with an HDR template. We also examine the utility of Nme2Cas9 target sites in the vector for self-inactivation. We demonstrate that these platforms can effectively treat two disease models [type I hereditary tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I)] in mice. These results will enable single-vector AAVs to achieve diverse therapeutic genome editing outcomes.

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Mycobacteria excises DNA damage in 12- or 13-nucleotide-long oligomers by prokaryotic-type dual incisions and performs transcription-coupled repair

Project description:In nucleotide excision repair, bulky DNA lesions such as UV-induced cyclobutane pyrimidine dimers (CPDs) are removed from the genome by concerted dual incisions bracketing the lesion, followed by gap filling and ligation. So far, two dual incision patterns have been discovered: prokaryotic type which removes the damage in 11-13 nucleotide-long oligomers and eukaryotic type which removes the damage in 24-32 nucleotide-long oligomers. However, a recent study reported that the UvrC protein of Mycobacterium tuberculosis removes damage in a manner analogous to yeast and humans in a 25-mer oligonucleotide arising from incisions at 15 nucleotides from the 3’ end and 9 nucleotides from the 5’ end flanking the damage. To test this model we used the in vivo excision assay and the XR-seq genome-wide repair mapping method developed in our lab to determine the repair pattern and genome-wide repair map of M. smegmatis. We find that M. smegmatis, which possesses homologues of the E. coli UvrA, B, and C genes, removes CPDs from the genome in a manner identical to the prokaryotic pattern by incising 7 nucleotides 5’ and 3 or 4 nucleotides 3’ to the photoproduct.

2020-10-06 | GSE159048 | GEO

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2024-04-01 | GSE253191 | GEO

Precise and broad scope genome editing based on high-specificity Cas9 nickases

Project description:RNA-guided nucleases (RGNs) based on CRISPR systems permit installing short and large edits within eukaryotic genomes. However, precise genome editing is often hindered due to nuclease off- target activities and the multiple-copy character of the vast majority of chromosomal sequences. Dual nicking RGNs and high-specificity RGNs both exhibit low off-target activities. Here, we report that high-specificity Cas9 nucleases are convertible into nicking Cas9D10A variants whose precision is superior to that of the commonly used Cas9D10A nickase. Dual nicking RGNs based on a selected group of these Cas9D10A variants can yield gene knockouts and gene knock-ins at frequencies similar to or higher than those achieved by their conventional counterparts. Moreover, high-specificity dual nicking RGNs are capable of distinguishing highly similar sequences by “tiptoeing” over pre-existing single base-pair polymorphisms. Finally, high-specificity RNA-guided nicking complexes generally preserve genomic integrity, as demonstrated by unbiased genome-wide high-throughput sequencing assays. Thus, in addition to substantially enlarging the Cas9 nickase toolkit, we demonstrate the feasibility in expanding the range and precision of genome editing procedures. The herein introduced tools and multi-tier high-specificity genome editing strategies might be particularly beneficial whenever predictability and/or safety of genetic manipulations are paramount.

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Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes

Project description:Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, which must withstand decades of DNA damage without replicating. This lack of knowledge limits the efficiency and precision of genome editing in clinically relevant cells. To address this, we used induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. We discovered that neurons can take weeks to fully resolve this damage, compared to just days in isogenic iPSCs. Furthermore, Cas9-treated neurons upregulated unexpected DNA repair genes, including factors canonically associated with replication. Manipulating this response with chemical or genetic perturbations allowed us to direct neuronal repair toward desired editing outcomes. By studying DNA repair in postmitotic human cells, we uncovered unforeseen challenges and opportunities for precise therapeutic editing.

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