Department of Stem Cell Biology, Centre of Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom .
Stem Cells Dev. 2018 Mar 15;27(6):391-404. doi: 10.1089/scd.2017.0268. Epub 2018 Mar 12.
Modeling disease with human pluripotent stem cells (hPSCs) is hindered because the impact on cell phenotype from genetic variability between individuals can be greater than from the pathogenic mutation. While "footprint-free" Cas9/CRISPR editing solves this issue, existing approaches are inefficient or lengthy. In this study, a simplified PiggyBac strategy shortened hPSC editing by 2 weeks and required one round of clonal expansion and genotyping rather than two, with similar efficiencies to the longer conventional process. Success was shown across four cardiac-associated loci (ADRB2, GRK5, RYR2, and ACTC1) by genomic cleavage and editing efficiencies of 8%-93% and 8%-67%, respectively, including mono- and/or biallelic events. Pluripotency was retained, as was differentiation into high-purity cardiomyocytes (CMs; 88%-99%). Using the GRK5 isogenic lines as an exemplar, chronic stimulation with the β-adrenoceptor agonist, isoprenaline, reduced beat rate in hPSC-CMs expressing GRK5-Q41 but not GRK5-L41; this was reversed by the β-blocker, propranolol. This shortened, footprint-free approach will be useful for mechanistic studies.
用人类多能干细胞（hPSC）建模疾病受到阻碍，因为个体间遗传变异对细胞表型的影响可能大于致病突变。虽然“无足迹”Cas9/CRISPR 编辑解决了这个问题，但现有的方法效率低下或耗时冗长。在这项研究中，简化的 PiggyBac 策略将 hPSC 编辑缩短了 2 周，并且只需要一轮克隆扩增和基因分型，而不是两轮，其效率与较长的传统过程相似。通过基因组切割和编辑效率分别为 8%-93%和 8%-67%，在四个与心脏相关的基因座（ADRB2、GRK5、RYR2 和 ACTC1）中都显示出了成功，包括单等位基因和/或双等位基因事件。多能性得以保留，分化为高纯度心肌细胞（CM）的效率也保持在 88%-99%。使用 GRK5 同基因系作为范例，β-肾上腺素受体激动剂异丙肾上腺素对表达 GRK5-Q41 的 hPSC-CM 的刺激会降低心率，但对表达 GRK5-L41 的 hPSC-CM 没有影响；这种影响可以被β受体阻滞剂普萘洛尔逆转。这种简化的、无足迹的方法将有助于进行机制研究。
