School of Biological and Health Systems Engineering, Arizona State University, 501 E. Tyler Mall, ECG 334A, Tempe, AZ 85287, USA; Graduate Program in Clinical Translational Sciences, University of Arizona College of Medicine-Phoenix, Phoenix, AZ 85004, USA.
School of Biological and Health Systems Engineering, Arizona State University, 501 E. Tyler Mall, ECG 334A, Tempe, AZ 85287, USA.
Stem Cell Reports. 2020 Feb 11;14(2):184-191. doi: 10.1016/j.stemcr.2019.12.013. Epub 2020 Jan 30.
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.
目前，在人类多能干细胞（hPSC）中进行 CRISPR 靶向的单核苷酸修饰和随后的同源定向修复（HDR）同型细胞系的产生，需要引入有害的双链 DNA 断裂，然后进行低效的 HDR。在这里，我们利用 Cas9 脱氨酶碱基编辑技术共同靶向基因组位点和一个附加体报告基因，以在不进行 HDR 的情况下在 hPSC 中实现单核苷酸基因组改变。这种名为“使用瞬时报告基因进行编辑富集的碱基编辑同型 hPSC 系生成（BIG-TREE）”的方法可在多个 hPSC 系中实现 >80%的单核苷酸编辑效率。此外，我们还表明，BIG-TREE 可通过引入过早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了 hPSC 的高效多重编辑。这种易于采用的方法将允许对 hPSC 进行精确和高效的碱基编辑，用于发育生物学、疾病建模、药物筛选和基于细胞的治疗。
