Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities

Abstract

Genome editing in pluripotent stem cells (PSCs) using CRISPR technology holds great promise for therapeutic applications. Yet, it has been reported that Cas9-mediated cleavage could cause large deletions or rearrangements of DNA, and the selection of edited PSCs could acquire p53 mutations. Adenine base editors (ABEs) do not introduce DNA double-strand breaks and thus have been proposed as alternatives to circumvent those problems, but their off-target effects still limit their applications. Here, we tested different combinations of off-target reduction methods to further diminish off-target effects of ABEs without compromising their on-target editing efficiencies. We subsequently chose the best editor, CE-8e-dV, which contains V106W substitution, R153 deletion, and Cas-embedding strategy, to establish a single-cell-derived human embryonic stem cell (hESC) line expressing tetracycline-inducible CE-8e-dV. By performing RNA and whole-genome sequencing, we demonstrated that the expression of CE-8e-dV did not produce nearly any DNA or RNA off-target effects in hESCs. Our results provide stringent proof of the safety of ABEs in PSCs and suggest that CE-8e-dV could be suitable for related therapeutic strategies, such as generation of engineered stem cells in vitro and gene therapy in vivo.