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Eliminating genome-wide and transcriptome-wide off-target mutations by base editor

ABSTRACT: Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications, but suffers from off-target (OT) mutations. By taking advantage of a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, a transformer BE (tBE) system is developed to induce efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, tBE remains inactive at OT sites with the fusion of a cleavable dCDI, thus eliminating unintended mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base changes. After delivery into mice via a dual-AAV system, tBE created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9 level, which resulted in ~30-40% decrease of total cholesterol. Together, the development of tBE establishes a highly-precise base editing system and its in vivo efficacy envisions potential therapeutic applications.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE164477 | GEO | 2021/03/12

REPOSITORIES: GEO

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Eliminating genome-wide and transcriptome-wide off-target mutations by base editor [tBE_target_genomic_seq]

Project description:Conjugation of CRISPR-Cas9 with cytidine deaminases leads to base editors (BEs) for programmable C-to-T editing, which holds potentials in clinical applications, but suffers from off-target (OT) mutations. By taking advantage of a cleavable deoxycytidine deaminase inhibitor (dCDI) domain, a transformer BE (tBE) system is developed to induce efficient editing with only background levels of genome-wide and transcriptome-wide OT mutations. After being produced, tBE remains inactive at OT sites with the fusion of a cleavable dCDI, thus eliminating unintended mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base changes. After delivery into mice via a dual-AAV system, tBE created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9 level, which resulted in ~30-40% decrease of total cholesterol. Together, the development of tBE establishes a highly-precise base editing system and its in vivo efficacy envisions potential therapeutic applications.

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