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Efficient precise integration of large DNA sequences with 3'-overhang dsDNA donors using CRISPR/Cas9.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2023 May 30; Vol. 120 (22), pp. e2221127120. Date of Electronic Publication: 2023 May 22. - Publication Year :
- 2023
-
Abstract
- 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 ( L ong dsDNA with 3'- O verhangs mediated C RISPR K nock-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.
Details
- Language :
- English
- ISSN :
- 1091-6490
- Volume :
- 120
- Issue :
- 22
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 37216515
- Full Text :
- https://doi.org/10.1073/pnas.2221127120