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A truncated reverse transcriptase enhances prime editing by split AAV vectors

ABSTRACT: A truncated reverse transcriptase enhances prime editing by split AAV vectors

PROVIDER: PRJNA846287 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR19544784_1.fastq.gz	Fastqsanger.gz
	SRR19544784_2.fastq.gz	Fastqsanger.gz
	SRR19544785_1.fastq.gz	Fastqsanger.gz
	SRR19544785_2.fastq.gz	Fastqsanger.gz
	SRR19544786_1.fastq.gz	Fastqsanger.gz

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A truncated reverse transcriptase enhances prime editing by split AAV vectors

Project description:To investigate if the truncated PE can be dilivered by dual AAV8 vectors for in vivo prime editing. We injected the dual AAV8 into 10-week-old C57BL/6J mice . Livers were isolated 4 weeks after injection and next generation sequencing showed an average of 1.4% and 5.4% precise prime editing with the low and high AAV doses, respectively (Figure 4D ). This demonstrates that PECO-Mini can be efficiently delivered by dual AAVs for in vivo prime editing.

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A flexible split prime editor using truncated reverse transcriptase improves dual-AAV delivery in mouse liver.

Project description:Prime editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9-which affects editing efficiency-is limited due to the large size of PE. Furthermore, overexpressing reverse transcriptase in mammalian cells might disrupt translation termination via its RNase H domain. Here, we developed a compact PE without the RNase H domain that showed editing comparable with full-length PE. With compact PE, we used a Cas9 split site (Glu 573) that supported robust editing in cells (up to 93% of full-length PE) and in mouse liver. We then demonstrated that split-cPE573 delivered by dual-AAV8 efficiently mediated a 3-bp TGA insertion in the Pcsk9 gene in mouse liver. Compact PE without the RNase H domain abolished its binding to peptidyl release factor 1 (eRF1) and mitigated the stop codon readthrough effect observed with full-length PE. This study identifies a compact PE with a flexible split design to advance utility of prime editing in vivo.

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