Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.
Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.
复制标题
DOI：
10.1073/pnas.2221127120
复制
发表时间：
2023-05-30
影响因子：
11.1
通讯作者：
Liang, Haojun
中科院分区：
文献类型：
--
作者：
Han, Wenjie;Li, Zhigang;Guo, Yijun;He, Kaining;Li, Wenqing;Xu, Caoling;Ge, Lishuang;He, Miao;Yin, Xue;Zhou, Junxiang;Li, Chengxu;Yao, Dongbao;Bao, Jianqiang;Liang, Haojun
关键词：
我们设计了一种实用、高效的方法，称为基因敲入法，该方法利用具有杂合“3′-突出端dsDNA”（odsDNA）结构的供体，高效、准确地敲入大片段DNA。这种给体可以容易地以低成本合成。此外，odsDNA供体可以直接或间接连接到Cas9融合蛋白和延伸的sgRNA，并实现2，500 bp基因的有效原位敲入。CRISPR/Cas9基因组编辑工具极大地提高了我们在生物医学研究和创新生物技术中操纵真核生物基因组的能力。然而，目前允许基因大小的大DNA片段的精确整合的方法通常效率低且成本高。在此，我们开发了一种通用且高效的方法，称为CRISPR敲入（Long dsDNA with 3′-Overhangs mediated CRISPR Knock-in），该方法利用专门设计的含有50-nt同源臂的3′-overhangs双链DNA（odsDNA）供体。odsDNA的3′-overhangs长度由5个连续的硫代磷酸酯修饰指定。与现有方法相比，该方法允许将内切酶大小的DNA片段高效靶向插入哺乳动物基因组中，成本低，脱靶效应低，敲入频率比传统的基于同源重组的方法高5倍以上。这种新设计的基于同源性定向修复的DNA修复方法是一种强大的工具，适用于基因工程、基因治疗和合成生物学迫切需要的基因大小的片段整合。
Here, we devised a practical and efficient method, termed LOCK, which utilizes a donor with hybrid “3′-overhang dsDNA” (odsDNA) structure for efficient and accurate knock-in of large DNA fragment. This donor could be readily synthesized with low cost. In addition, the odsDNA donor could be directly or indirectly attached to Cas9 fusion protein and extended sgRNA and achieve efficient knock-in of 2,500 bp genes in situ. CRISPR/Cas9 genome-editing tools have tremendously boosted our capability of manipulating the eukaryotic genomes in biomedical research and innovative biotechnologies. However, the current approaches that allow precise integration of gene-sized large DNA fragments generally suffer from low efficiency and high cost. Herein, we developed a versatile and efficient approach, termed LOCK (Long dsDNA with 3′-Overhangs mediated CRISPR Knock-in), by utilizing specially designed 3′-overhang double-stranded DNA (odsDNA) donors harboring 50-nt homology arm. The length of the 3′-overhangs of odsDNA is specified by the five consecutive phosphorothioate modifications. Compared with existing methods, LOCK allows highly efficient targeted insertion of kilobase-sized DNA fragments into the mammalian genomes with low cost and low off-target effects, yielding >fivefold higher knock-in frequencies than conventional homologous recombination-based approaches. This newly designed LOCK approach based on homology-directed repair is a powerful tool suitable for gene-sized fragment integration that is urgently needed for genetic engineering, gene therapies, and synthetic biology.
登录
查看更多内容
影响因子：
14.9
作者：
Brinkman, Eva K.;Kousholt, Arne N.;van Steensel, Bas
通讯作者：
van Steensel, Bas
影响因子：
4.1
作者：
Liang, Xiquan;Potter, Jason;Chesnut, Jonathan D.
通讯作者：
Chesnut, Jonathan D.
影响因子：
7
作者：
Kan Y;Ruis B;Takasugi T;Hendrickson EA
通讯作者：
Hendrickson EA
影响因子：
7.7
作者：
Cruz-Becerra, Grisel;Kadonaga, James T.
通讯作者：
Kadonaga, James T.
影响因子：
7.7
作者：
Gutierrez-Triana JA;Tavhelidse T;Thumberger T;Thomas I;Wittbrodt B;Kellner T;Anlas K;Tsingos E;Wittbrodt J
通讯作者：
Wittbrodt J