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Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons [PRAISE]

ABSTRACT: Nonsense mutations, responsible for ~11% of genetic diseases, create premature stop codons (PTCs) and lead to truncated and often non-functional proteins. Recently, programmable RNA pseudouridylation has emerged as a new type of RNA base editor to suppress PTCs. However, current methods suffer from low efficiency and limited precision. Here, we develop RESTART v3, an updated version of RESTART, which utilizes near-cognate tRNAs to improve the readthrough efficiency of pseudouridine-modified PTCs. We show an average of ~5-fold higher editing efficiency than RESTART v2 in cultured cells, currently the most active RNA pseudouridylation tool to mediate PTC readthrough. Moreover, RESTART v3 achieves functional PTC readthrough in disease cell models of cystic fibrosis and Hurler syndrome. Furthermore, RESTART v3 enables accurate incorporation of the original amino acid for nearly half of the PTC sites, considering the naturally occurring frequencies of sense to nonsense codons. In line with the benign off-target effect of RESTART, RESTART v3 does not change the coding information nor the expression level of transcripts with off-target editing; except for the specifically overexpressed tRNA molecule, it does not alter the expression level of endogenous tRNA pool. Overall, RESTART v3 represents an enhanced RNA base editor with increased efficiency and accuracy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE237632 | GEO | 2024/01/29

REPOSITORIES: GEO

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Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons [tRNA-seq]

Project description:Nonsense mutations, responsible for ~11% of genetic diseases, create premature stop codons (PTCs) and lead to truncated and often non-functional proteins. Recently, programmable RNA pseudouridylation has emerged as a new type of RNA base editor to suppress PTCs. However, current methods suffer from low efficiency and limited precision. Here, we develop RESTART v3, an updated version of RESTART, which utilizes near-cognate tRNAs to improve the readthrough efficiency of pseudouridine-modified PTCs. We show an average of ~5-fold higher editing efficiency than RESTART v2 in cultured cells, currently the most active RNA pseudouridylation tool to mediate PTC readthrough. Moreover, RESTART v3 achieves functional PTC readthrough in disease cell models of cystic fibrosis and Hurler syndrome. Furthermore, RESTART v3 enables accurate incorporation of the original amino acid for nearly half of the PTC sites, considering the naturally occurring frequencies of sense to nonsense codons. In line with the benign off-target effect of RESTART, RESTART v3 does not change the coding information nor the expression level of transcripts with off-target editing; except for the specifically overexpressed tRNA molecule, it does not alter the expression level of endogenous tRNA pool. Overall, RESTART v3 represents an enhanced RNA base editor with increased efficiency and accuracy.

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