The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigate the potential off-target structural variations associated with CRISPR/Cas9, ABE, and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. The results show that both Cas9 and ABE generate off-target structural variations (SVs) in mouse embryos, while CBE induces rare SVs. In addition, off-target large deletions are detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activate the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulate cell cycle-related genes and are arrested in the G0 phase. A percentage of 16.59% and 4.29% aneuploid cells are still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE are universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs are significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). This study shows both CRISPR/Cas9 and ABE induce off-target SVs in mouse embryos and primary human T cells, raising an urgent need for the development of high-fidelity gene editing tools.

中文翻译：

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腺嘌呤碱基编辑器在小鼠胚胎和原代人 T 细胞中诱导脱靶结构变化


基于 CRISPR 的基因编辑方法的安全性在临床应用中是重中之重。先前的研究报道，Cas9 切割在原代人 T 细胞中诱导了频繁的非整倍体，但切割介导的碱基编辑器编辑是否会产生脱靶结构变化仍然未知。在这里，我们通过全基因组测序和单细胞 RNA-seq 分析研究了小鼠胚胎和原代人类 T 细胞中与 CRISPR/Cas9、ABE 和 CBE 编辑相关的潜在脱靶结构变异。结果表明，Cas9 和 ABE 都在小鼠胚胎中产生脱靶结构变异 （SV），而 CBE 诱导罕见的 SV。此外，在转染 Cas9 的 32.74% 的原代人 T 细胞和 9.17% 的转染 ABE 的细胞中检测到脱靶大缺失。此外，Cas9 诱导的非整倍体细胞激活 P53 和细胞凋亡途径，而 ABE 相关的非整倍体细胞显著上调细胞周期相关基因并停滞在 G0 期。在转染 Cas9 或 ABE 后 3 周仍可观察到 16.59% 和 4.29% 非整倍体细胞的百分比。ABE 中的这些脱靶现象在其他细胞类型（如 B 细胞和 Huh7）中观察到是普遍的。此外，在用高保真 ABE （ABE-V106W） 处理的细胞中，脱靶 SV 显着降低。这项研究表明，CRISPR/Cas9 和 ABE 都会在小鼠胚胎和原代人类 T 细胞中诱导脱靶 SVs，这引发了对开发高保真基因编辑工具的迫切需求。