Applying programmable nucleases in gene editing has greatly shaped current research in basic biology and clinical translation. Gene editing in human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), is highly relevant to clinical cell therapy and thus should be examined with particular caution. First, since all mutations in PSCs will be carried to all their progenies, off-target edits of editors will be amplified. Second, due to the hypersensitivity of PSCs to DNA damage, double-strand breaks (DSBs) made by gene editing could lead to low editing efficiency and the enrichment of cell populations with defective genomic safeguards. In this regard, DSB-independent gene editing tools, such as base editors and prime editors, are favored due to their nature to avoid these consequences. With more understanding of the microbial world, new systems, such as Cas-related nucleases, transposons, and recombinases, are also expanding the toolbox for gene editing. In this review, we discuss current applications of programmable nucleases in PSCs for gene editing, the efforts researchers have made to optimize these systems, as well as new tools that can be potentially employed for differentiation modeling and therapeutic applications.

Early embryonic development in mammals, from fertilization to implantation, can be viewed as a process in which stem cells alternate between self-renewal and differentiation. During this process, the fates of stem cells in embryos are gradually specified, from the totipotent state, through the segregation of embryonic and extraembryonic lineages, to the molecular and cellular defined progenitors. Most of those stem cells with different potencies in vivo can be propagated in vitro and recapitulate their differentiation abilities. Complex and coordinated regulations, such as epigenetic reprogramming, maternal RNA clearance, transcriptional and translational landscape changes, as well as the signal transduction, are required for the proper development of early embryos. Accumulated studies suggest that Dicer-dependent noncoding RNAs, including microRNAs (miRNAs) and endogenous small-interfering RNAs (endo-siRNAs), are involved in those regulations and therefore modulate biological properties of stem cells in vitro and in vivo. Elucidating roles of these noncoding RNAs will give us a more comprehensive picture of mammalian embryonic development and enable us to modulate stem cell potencies. In this review, we will discuss roles of miRNAs in regulating the maintenance and cell fate potential of stem cells in/from mouse and human early embryos.

The authors wish to make the following correction to this paper