التفاصيل البيبلوغرافية
العنوان: |
Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9. |
المؤلفون: |
Wenjie Han, Zhigang Li, Yijun Guo, Kaining He, Wenqing Li, Caoling Xu, Lishuang Ge, Miao He, Xue Yin, Junxiang Zhou, Chengxu Li, Dongbao Yao, Jianqiang Bao, Haojun Liang |
المصدر: |
Proceedings of the National Academy of Sciences of the United States of America; 5/30/2023, Vol. 120 Issue 22, p1-11, 49p |
مصطلحات موضوعية: |
GENETIC engineering, GENOMICS, CRISPRS, DNA sequencing, EUKARYOTIC genomes |
مستخلص: |
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. [ABSTRACT FROM AUTHOR] |
|
Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) |
قاعدة البيانات: |
Complementary Index |