Laboratory of Clinical Chemistry, Sahlgrenska University Hospital, Gothenburg, Sweden.
Stem Cells and Genome Editing, Genomics and Cellular Sciences, Agriculture Victoria Research, Bundoora, VIC, Australia.
Methods Mol Biol. 2022;2495:29-46. doi: 10.1007/978-1-0716-2301-5_2.
The last two decades have marked significant advancement in the genome editing field. Three generations of programmable nucleases (ZFNs, TALENs, and CRISPR-Cas system) have been adopted to introduce targeted DNA double-strand breaks (DSBs) in eukaryotic cells. DNA repair machinery of the cells has been exploited to introduce insertion and deletions (indels) at the targeted DSBs to study function of any gene-of-interest. The resulting indels were generally assumed to be "random" events produced by "error-prone" DNA repair pathways. However, recent advances in computational tools developed to study the Cas9-induced mutations have changed the consensus and implied the "non-randomness" nature of these mutations. Furthermore, CRISPR-centric tools are evolving at an unprecedented pace, for example, base- and prime-editors are the newest developments that have been added to the genome editing toolbox. Altogether, genome editing tools have revolutionized our way of conducting research in life sciences. Here, we present a concise overview of genome editing tools and describe the DNA repair pathways underlying the generation of genome editing outcome.
过去的二十年见证了基因组编辑领域的重大进展。已经采用了三代可编程核酸酶(锌指核酸酶、转录激活因子样效应物核酸酶和 CRISPR-Cas 系统)在真核细胞中引入靶向 DNA 双链断裂(DSB)。细胞的 DNA 修复机制被用来在靶向 DSB 处引入插入和缺失(indels),以研究任何感兴趣基因的功能。产生的 indels 通常被认为是由“易错”DNA 修复途径产生的“随机”事件。然而,最近开发的用于研究 Cas9 诱导突变的计算工具的进步改变了这一共识,并暗示了这些突变的“非随机性”本质。此外,以 CRISPR 为中心的工具正在以前所未有的速度发展,例如,碱基和引物编辑器是最近添加到基因组编辑工具包中的最新发展。总之,基因组编辑工具彻底改变了我们在生命科学领域开展研究的方式。在这里,我们简要概述了基因组编辑工具,并描述了产生基因组编辑结果的 DNA 修复途径。