Isogenic induced pluripotent stem cell (iPSC) lines are currently mostly created by homology directed repair evoked by a double-strand break (DSB) generated by CRISPR-Cas9. However, this process is in general lengthy and inefficient. This problem can be overcome, specifically for correction or insertion of transition mutations, by using base editing (BE). BE does not require DSB formation, hence avoiding creation of genomic off-target breaks and insertions and deletions, and as it is highly efficient, it also does not require integration of selection cassettes in the genome to enrich for edited cells. BE has been successfully used in many cell types as well as in some in vivo settings to correct or insert mutations, but very few studies have reported generation of isogenic iPSC lines using BE. Here, we describe a simple and fast workflow to generate isogenic iPSCs efficiently with a compound heterozygous or a homozygous Wolfram syndrome 1 (WFS1) mutation using adenine BE, without the need to include a genomic selection cassette and without off-target modifications. We demonstrated that correctly base-edited clones can be generated by screening only five cell clones in less than a month, provided that the mutation is positioned in a correct place with regards to the protospacer adjacent motif sequence and no putative bystander bases exist.

中文翻译：

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使用腺嘌呤碱基编辑快速高效地生成同基因诱导多能干细胞系


目前，同基因诱导多能干细胞 (iPSC) 系主要是通过 CRISPR-Cas9 产生的双链断裂 (DSB) 引发的同源定向修复来创建的。然而，这个过程通常是漫长且低效的。这个问题可以通过使用碱基编辑（BE）来克服，特别是对于过渡突变的校正或插入。 BE 不需要 DSB 形成，因此避免了基因组脱靶断裂以及插入和删除的产生，并且由于它非常高效，因此它也不需要在基因组中整合选择盒来富集编辑的细胞。 BE 已成功用于许多细胞类型以及一些体内环境中以纠正或插入突变，但很少有研究报道使用 BE 生成同基因 iPSC 系。在这里，我们描述了一个简单而快速的工作流程，可使用腺嘌呤 BE 高效生成具有复合杂合或纯合 Wolfram 综合征 1 ( WFS1 ) 突变的同基因 iPSC，无需包含基因组选择盒，也无需脱靶修饰。我们证明，只要突变位于原型间隔子相邻基序序列的正确位置并且不存在假定的旁观者碱基，则可以通过在不到一个月的时间内筛选五个细胞克隆来生成正确的碱基编辑克隆。