Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea.
National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Bac Tu Liem, Hanoi, Vietnam.
Biotechnol J. 2022 Jul;17(7):e2100413. doi: 10.1002/biot.202100413. Epub 2021 Dec 10.
Spontaneous double-stranded DNA breaks (DSBs) frequently occur within the genome of all living organisms and must be well repaired for survival. Recently, more important roles of the DSB repair pathways that were previously thought to be minor pathways, such as single-strand annealing (SSA), have been shown. Nevertheless, the biochemical mechanisms and applications of the SSA pathway in genome editing have not been updated.
Understanding the molecular mechanism of SSA is important to design potential applications in gene editing. This review provides insights into the recent progress of SSA studies and establishes a model for their potential applications in precision genome editing.
The SSA mechanism involved in DNA DSB repair appears to be activated by a complex signaling cascade starting with broken end sensing and 5'-3' resection to reveal homologous repeats on the 3' ssDNA overhangs that flank the DSB. Annealing the repeats would help to amend the discontinuous ends and restore the intact genome, resulting in the missing of one repeat and the intervening sequence between the repeats. We proposed a model for CRISPR-Cas-based precision insertion or replacement of DNA fragments to take advantage of the characteristics. The proposed model can add a tool to extend the choice for precision gene editing. Nevertheless, the model needs to be experimentally validated and optimized with SSA-favorable conditions for practical applications.
在所有生物的基因组中，自发的双链 DNA 断裂（DSBs）经常发生，为了生存，这些断裂必须得到很好的修复。最近，以前被认为是次要途径的 DSB 修复途径，如单链退火（SSA），其重要性作用得到了更多的展示。然而，SSA 途径在基因组编辑中的生化机制和应用尚未得到更新。
了解 SSA 的分子机制对于设计潜在的基因编辑应用很重要。这篇综述提供了对 SSA 研究的最新进展的深入了解，并为其在精确基因组编辑中的潜在应用建立了一个模型。
涉及 DNA DSB 修复的 SSA 机制似乎是通过一个复杂的信号级联反应激活的，该反应从断裂末端的感应和 5' - 3' 切除开始，以揭示 DSB 侧翼的 3' ssDNA 突出端上的同源重复序列。重复退火有助于修正不连续的末端并恢复完整的基因组，导致一个重复缺失和重复之间的插入序列缺失。我们提出了一个基于 CRISPR-Cas 的精确插入或替换 DNA 片段的模型，以利用这些特征。该模型可以为精确基因编辑增加一个选择工具。然而，该模型需要通过实验验证和优化，以获得有利于 SSA 的条件，从而实现实际应用。
