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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
	SRR26808084_1.fastq.gz	Fastqsanger.gz
	SRR26808084_2.fastq.gz	Fastqsanger.gz
	SRR26808085_1.fastq.gz	Fastqsanger.gz
	SRR26808085_2.fastq.gz	Fastqsanger.gz
	SRR26808086_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 [RNA-seq]

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.

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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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