Abstract
Precise gene editing is—or will soon be—in clinical use for several diseases, and more applications are under development. The programmable nuclease Cas9, directed by a single-guide RNA (sgRNA), can introduce double-strand breaks (DSBs) in target sites of genomic DNA, which constitutes the initial step of gene editing using this novel technology. In mammals, two pathways dominate the repair of the DSBs—nonhomologous end joining (NHEJ) and homology-directed repair (HDR)—and the outcome of gene editing mainly depends on the choice between these two repair pathways. Although HDR is attractive for its high fidelity, the choice of repair pathway is biased in a biological context. Mammalian cells preferentially employ NHEJ over HDR through several mechanisms: NHEJ is active throughout the cell cycle, whereas HDR is restricted to S/G2 phases; NHEJ is faster than HDR; and NHEJ suppresses the HDR process. This suggests that definitive control of outcome of the programmed DNA lesioning could be achieved through manipulating the choice of cellular repair pathway. In this review, we summarize the DSB repair pathways, the mechanisms involved in choice selection based on DNA resection, and make progress in the research investigating strategies that favor Cas9-mediated HDR based on the manipulation of repair pathway choice to increase the frequency of HDR in mammalian cells. The remaining problems in improving HDR efficiency are also discussed. This review should facilitate the development of CRISPR/Cas9 technology to achieve more precise gene editing.
Highlights
Precise genomic editing based on programming nucleases is opening up the possibility of achieving desired genome editing outcome in vitro and in vivo, and ensure its safe and rapid adoption for genome engineering applications across biology [1,2]
The frequency of homology-directed repair (HDR) in nature is extremely low, and mammalian cells preferentially employ nonhomologous end joining (NHEJ) over HDR through several mechanisms: NHEJ is active throughout the cell cycle except in mitosis, whereas HDR is restricted to S and G2 phases; NHEJ is faster than HDR; and NHEJ represses HDR through a series of mechanisms [16]
Owing to the role of KU in promoting NHEJ and suppressing HDR, Li et al used KU-specific siRNA to downregulate the expression of Ku70 and Ku80, which resulted in a significant increase in the frequency of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated HDR in pig fibroblasts [94]
Summary
Precise genomic editing based on programming nucleases is opening up the possibility of achieving desired genome editing outcome in vitro and in vivo, and ensure its safe and rapid adoption for genome engineering applications across biology [1,2]. The discovery of zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) has greatly improved genome editing efficacy, using these systems for gene editing at new target sites in the genome requires re-designing or re-engineering a new set of proteins, which restricts their broad application [3]. The major step committing a DSB to HDR is 5 -to-3 resection of the DNA end to form a 3 single-stranded DNA overhang [21] This process is initiated by the MRN (MRE11-RAD50-NBS1) complex, which serves as a scaffold for amplifying the ATM signaling response to the DSB [22]. DNA bases are randomly added and removed through the resulting in small indels relative to the original genomic template, which constitutes the basis of NHEJ-based error-prone editing (Figure 2)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
