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DNA repair-induced mutagenesis

APOBEC3 can trigger cytidine deamination in single-stranded oligodeoxynucleotides, which ultimately results in base substitution mutations in genomic DNA through homology-directed repair (HDR) of Cas9-generated double-strand breaks. In addition, the APOBEC3-catalyzed deamination in the genomic single-stranded DNA formed during the repair of Cas9 nickase-generated single-strand breaks in human cells can be further processed to yield mutations mainly involving insertions or deletions (indels). Both APOBEC3-mediated deamination and DNA-repair proteins play important roles in the generation of these indels. Therefore, optimizing conditions for the repair of CRISPR–Cas9-generated DNA breaks, such as using double-stranded donors in HDR or temporarily suppressing endogenous APOBEC3s, can repress these unwanted mutations in genomic DNA.

Development of base editor

Though base editors (BEs) are efficient in inducing targeted base editing, BEs can also trigger both genome-wide and transcriptome-wide off-target mutations. Importantly, off-target mutations can occur at the sites with no sequence similarity to sgRNA and thus cannot be predicted, which impedes BE's applications, particularly for therapeutics. By taking advantage of deoxycytidine deaminase inhibitor (dCDI) domains and split-TEV-protease, we developed a novel transformer base editor (tBE) system. The tBE system remains inactive at off-target sites with a cleavable fusion of dCDI domain, thus eliminating off-target mutations. Only when binding at on-target sites, tBE is transformed to cleave off the dCDI domain and catalyzes targeted deamination for precise base editing.

Gene editing therapy

Ex vivo editing: β-hemoglobinopathies, including β-thalassemia and sickle cell disease (SCD), are the common genetic diseases caused by mutations in the hemoglobin subunit β (HBB) gene locus. Comparing to Cas9 nuclease or conventional BEs (e.g., ABE8e and hA3A-BE3), we found that tBE-mediated disruption of the BCL11A-binding motif in the HBG1/2 promoters triggered high levels of hemoglobin production in hematopoietic stem cells from healthy donors and β-thalassemia patients while exhibiting no detectable DNA or RNA off-target mutations.

In vivo editing: Adeno-associated virus (AAV) is an efficient and widely used delivery agent, which however has a genome packaging size limit of ≤ 4.7 kb. The size of the conventional base editors precludes packaging in a single AAV vector. However, the tBE system is composed of small components, which could be conveniently packaged into a dual-AAV system to maintain editing efficiency maximally. We delivered the tBE system into mice through a dual-AAV system, which created a premature stop codon in Pcsk9 and significantly reduced serum PCSK9, resulting in a ~30-40% decrease in total cholesterol.

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