Abstract
The groundbreaking CRISPR technology is revolutionizing biomedical research with its superior simplicity, high efficiency, and robust accuracy. Recent technological advances by a coupling CRISPR system with various DNA repair mechanisms have further opened up new opportunities to overcome existing challenges in knocking-in foreign DNA in human pluripotent stem cells, including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). In this review, we summarized the very recent development of CRISPR-based knock-in strategies and discussed the results obtained as well as potential applications in human ESC and iPSC.
Highlights
Successful isolation of embryonic stem cells (ESC) and reprogramming of somatic tissues into induced pluripotent stem cells significantly foster the stem cell research and development of regenerative medicine [1]
To harness the full application potentials of human ESC/induced pluripotent stem cells (iPSC), targeted genome editing with high accuracy and efficiency has long been thought desirable
Back in the late 1980s, targeted genome editing through homologous recombination (HR) was first established in mouse ESC and demonstrated in generating live mouse strains carrying predesigned genetic modification [2]
Summary
Successful isolation of embryonic stem cells (ESC) and reprogramming of somatic tissues into induced pluripotent stem cells (iPSC) significantly foster the stem cell research and development of regenerative medicine [1]. With the application of engineered nucleases, DSBs are induced at selected target sites and trigger various DNA repair processes, via the homology-directed repair (HDR) (termed HR previously), the nonhomologous end joining (NHEJ), or the recently identified microhomologymediated end joining (MMEJ) pathways [10, 11] Studies have exploited these diverse DNA repair mechanisms to develop various targeting strategies and introduce a broad range of genomic modifications [12, 13]. With a step further, long single-strand DNA (lssDNA) has been employed for exogenous DNA knock-in through zygote injection [34] This lssDNAbased knock-in demonstrated higher targeting efficiency than traditional HDR-based methods and is more suitable to generate large-scale Cre-LoxP animal resources [35]. It is interesting but remains to be confirmed whether the lssDNA-mediated or the ssODN-facilitated dsDNAmediated HDR-based approaches are suitable for knock-in of large DNAs in human ESC/iPSC
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