Base editing is a form of genome editing that can directly convert a single base (C or A) to another base (T or G), which is of great potential in biomedical applications. The broad application of base editing is limited by its low activity and specificity, which still needs to be resolved. To address this, a simple and quick method for the determination of its activity/specificity is highly desired. Here, we developed a novel system, which could be harnessed for quick detection of editing activity and specificity of base editors (BEs) in human cells. Specifically, multiple cloning sites (MCS) were inserted into the human genome via lentivirus, and base editing targeting the MCS was performed with BEs. The base editing activities were assessed by specific restriction enzymes. The whole process only includes nucleotide-based targeting the MCS, editing, PCR, and digestion, thus, we named it NOTEPAD. This straightforward approach could be easily accessed by molecular biology laboratories. With this method, we could easily determine the BEs editing efficiency and pattern. The results revealed that BEs triggered more off-target effects in the genome than on plasmids including genomic indels (insertions and deletions). We found that ABEs (adenine base editors) had better fidelity than CBEs (cytosine base editors). Our system could be harnessed as a base editing assessment platform, which would pave the way for the development of next-generation BEs.

碱基编辑是基因组编辑的一种形式，可以将一个碱基（C或A）直接转换为另一个碱基（T或G），这在生物医学应用中具有巨大的潜力。基础编辑的广泛应用受到其低活性和专一性的限制，这仍然需要解决。为了解决这个问题，非常需要一种确定其活性/特异性的简单快捷的方法。在这里，我们开发了一种新颖的系统，可以利用该系统快速检测人类细胞中编辑活动和基础编辑器（BE）的特异性。具体地，通过慢病毒将多个克隆位点（MCS）插入人基因组，并用BE进行靶向MCS的碱基编辑。通过特定的限制酶评估碱基编辑活性。整个过程仅包括基于核苷酸的MCS定位，编辑，PCR和消化，因此，我们将其命名为NOTEPAD。分子生物学实验室可以很容易地获得这种简单的方法。使用这种方法，我们可以轻松确定BE的编辑效率和模式。结果表明，与在包括基因组插入/缺失（插入和缺失）的质粒上相比，BEs在基因组中引发了更多的脱靶效应。我们发现ABE（腺嘌呤碱基编辑）比CBE（胞嘧啶碱基编辑）具有更好的保真度。我们的系统可以用作基础编辑评估平台，这将为下一代BE的开发铺平道路。结果表明，与在包括基因组插入/缺失（插入和缺失）的质粒上相比，BEs在基因组中引发了更多的脱靶效应。我们发现ABE（腺嘌呤碱基编辑）比CBE（胞嘧啶碱基编辑）具有更好的保真度。我们的系统可以用作基础编辑评估平台，这将为下一代BE的开发铺平道路。结果表明，与在包括基因组插入/缺失（插入和缺失）的质粒上相比，BEs在基因组中引发了更多的脱靶效应。我们发现ABE（腺嘌呤碱基编辑）比CBE（胞嘧啶碱基编辑）具有更好的保真度。我们的系统可以用作基础编辑评估平台，这将为下一代BE的开发铺平道路。