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] |
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| قاعدة البيانات: |
Complementary Index |
