Park Ju-Chan, Jang Hyeon-Ki, Kim Jumee, Han Jun Hee, Jung Youngri, Kim Keuntae, Bae Sangsu, Cha Hyuk-Jin
College of Pharmacy, Seoul National University, 1, Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea.
Institute for Convergence of Basic Sciences, Hanyang University, Seoul 04763, Republic of Korea.
Mol Ther Nucleic Acids. 2021 Nov 29;27:175-183. doi: 10.1016/j.omtn.2021.11.023. eCollection 2022 Mar 8.
Precise genome editing of human pluripotent stem cells (hPSCs) is crucial not only for basic science but also for biomedical applications such as stem cell therapy and genetic disease modeling. However, hPSCs have unique cellular properties compared to somatic cells. For instance, hPSCs are extremely susceptible to DNA damage, and therefore Cas9-mediated DNA double-strand breaks (DSB) induce p53-dependent cell death, resulting in low Cas9 editing efficiency. Unlike Cas9 nucleases, base editors including cytosine base editor (CBE) and adenine base editor (ABE) can efficiently substitute single nucleotides without generating DSBs at target sites. Here, we found that the editing efficiency of CBE was significantly lower than that of ABE in human embryonic stem cells (hESCs), which are associated with high expression of DNA glycosylases, the key component of the base excision repair pathway. Sequential depletion of DNA glycosylases revealed that high expression of uracil DNA glycosylase (UNG) not only resulted in low editing efficiency but also affected CBE product purity (i.e., C to T) in hESCs. Therefore, additional suppression of UNG via transient knockdown would also improve C to T base substitutions in hESCs. These data suggest that the unique cellular characteristics of hPSCs could determine the efficiency of precise genome editing.
对人类多能干细胞（hPSC）进行精确的基因组编辑不仅对基础科学至关重要，而且对诸如干细胞治疗和遗传疾病建模等生物医学应用也至关重要。然而，与体细胞相比，hPSC具有独特的细胞特性。例如，hPSC极易受到DNA损伤，因此Cas9介导的DNA双链断裂（DSB）会诱导p53依赖性细胞死亡，导致Cas9编辑效率低下。与Cas9核酸酶不同，包括胞嘧啶碱基编辑器（CBE）和腺嘌呤碱基编辑器（ABE）在内的碱基编辑器可以有效替代单个核苷酸，而不会在靶位点产生DSB。在这里，我们发现，在人类胚胎干细胞（hESC）中，CBE的编辑效率明显低于ABE，这与碱基切除修复途径的关键成分DNA糖基化酶的高表达有关。对DNA糖基化酶的顺序缺失分析表明，尿嘧啶DNA糖基化酶（UNG）的高表达不仅导致编辑效率低下，而且还影响了hESC中CBE产物的纯度（即C到T）。因此，通过瞬时敲低对UNG进行额外抑制也将提高hESC中C到T的碱基替换效率。这些数据表明，hPSC独特的细胞特性可能决定精确基因组编辑的效率。
