Current CRISPR-targeted single-nucleotide modifications and subsequent isogenic cell line generation in human pluripotent stem cells (hPSCs) require the introduction of deleterious double-stranded DNA breaks followed by inefficient homology-directed repair (HDR). Here, we utilize Cas9 deaminase base-editing technologies to co-target genomic loci and an episomal reporter to enable single-nucleotide genomic changes in hPSCs without HDR. Together, this method entitled base-edited isogenic hPSC line generation using a transient reporter for editing enrichment (BIG-TREE) allows for single-nucleotide editing efficiencies of >80% across multiple hPSC lines. In addition, we show that BIG-TREE allows for efficient generation of loss-of-function hPSC lines via introduction of premature stop codons. Finally, we use BIG-TREE to achieve efficient multiplex editing of hPSCs at several independent loci. This easily adoptable method will allow for the precise and efficient base editing of hPSCs for use in developmental biology, disease modeling, drug screening, and cell-based therapies.

当前针对CRISPR的单核苷酸修饰以及随后在人多能干细胞（hPSC）中的同基因细胞系生成需要引入有害的双链DNA断裂，然后进行无效的同源性定向修复（HDR）。在这里，我们利用Cas9脱氨酶碱基编辑技术来共同靶向基因组基因座和附加型报告基因，以使hPSC中的单核苷酸基因组发生变化而无需HDR。在一起，这种方法称为碱基编辑的等基因hPSC系生成，使用瞬时报告基因进行编辑富集（BIG-TREE），可在多个hPSC系中实现> 80％的单核苷酸编辑效率。此外，我们表明BIG-TREE允许通过引入过早的终止密码子来有效生成功能丧失的hPSC系。最后，我们使用BIG-TREE在几个独立的基因座上实现hPSC的高效多路编辑。这种易于采用的方法将允许对hPSC进行精确有效的碱基编辑，以用于发育生物学，疾病建模，药物筛选和基于细胞的疗法。