A CRISPR/Cas-Mediated Selection-free Knockin Strategy in Human Embryonic Stem Cells

作者：Zhu Zengrong; Verma Nipun; Gonzalez Federico; Shi Zhong Dong; Huangfu Danwei^*

来源：Stem Cell Reports, 2015, 4(6): 1103-1111.

DOI：10.1016/j.stemcr.2015.04.016

The development of new gene-editing tools, in particular the CRISPR/Cas system, has greatly facilitated site-specific mutagenesis in human embryonic stem cells (hESCs), including the introduction or correction of patient-specific mutations for disease modeling. However, integration of a reporter gene into an endogenous locus in hESCs still requires a lengthy and laborious two-step strategy that involves first drug selection to identify correctly targeted clones and then excision of the drug-resistance cassette. Through the use of iCRISPR, an efficient gene-editing platform we recently developed, this study demonstrates a knockin strategy without drug selection for both active and silent genes in hESCs. Lineage-specific hESC reporter lines are useful for real-time monitoring of cell-fate decisions and lineage tracing, as well as enrichment of specific cell populations during hESC differentiation. Thus, this selection-free knockin strategy is expected to greatly facilitate the use of hESCs for developmental studies, disease modeling, and cell-replacement therapy.

• 出版日期2015-6-9