Chen Xinwen, Du Jingjing, Yun Shaowei, Xue Chaoyou, Yao Yao, Rao Shuquan
State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, China.
Tianjin Institutes of Health Science, Tianjin 301600, China.
Mol Ther Nucleic Acids. 2024 Feb 5;35(1):102138. doi: 10.1016/j.omtn.2024.102138. eCollection 2024 Mar 12.
Programmable genome insertion (or knock-in) is vital for both fundamental and translational research. The continuously expanding number of CRISPR-based genome insertion strategies demonstrates the ongoing development in this field. Common methods for site-specific genome insertion rely on cellular double-strand breaks repair pathways, such as homology-directed repair, non-homologous end-joining, and microhomology-mediated end joining. Recent advancements have further expanded the toolbox of programmable genome insertion techniques, including prime editing, integrase coupled with programmable nuclease, and CRISPR-associated transposon. These tools possess their own capabilities and limitations, promoting tremendous efforts to enhance editing efficiency, broaden targeting scope and improve editing specificity. In this review, we first summarize recent advances in programmable genome insertion techniques. We then elaborate on the cons and pros of each technique to assist researchers in making informed choices when using these tools. Finally, we identify opportunities for future improvements and applications in basic research and therapeutics.
可编程基因组插入（或敲入）对于基础研究和转化研究都至关重要。基于CRISPR的基因组插入策略数量不断增加，表明该领域正在不断发展。位点特异性基因组插入的常用方法依赖于细胞双链断裂修复途径，如同源定向修复、非同源末端连接和微同源性介导的末端连接。最近的进展进一步扩展了可编程基因组插入技术的工具库，包括碱基编辑、整合酶与可编程核酸酶结合以及CRISPR相关转座子。这些工具各有其能力和局限性，促使人们做出巨大努力来提高编辑效率、拓宽靶向范围并提高编辑特异性。在本综述中，我们首先总结可编程基因组插入技术的最新进展。然后详细阐述每种技术的优缺点，以帮助研究人员在使用这些工具时做出明智的选择。最后，我们确定了基础研究和治疗学未来改进和应用的机会。
