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| Status |
Public on Dec 01, 2017 |
| Title |
Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks |
| Organism |
Homo sapiens |
| Experiment type |
Other
|
| Summary |
The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only ~35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.
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| Overall design |
The genomic lamin locus is probed for template switching between two donor repair templates after repair of a Cas9-induced DNA break
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| Contributor(s) |
Paix A, Folkmann A, Goldman D, Kulaga H, Grzelak M, Rasoloson D, Paidemarry S, Green R, Reed R, Seydoux G |
| Citation(s) |
29183983 |
| Submission date |
Nov 30, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Daniel Goldman |
| E-mail(s) |
goldman@jhmi.edu
|
| Organization name |
Johns Hopkins School of Medicine
|
| Department |
Molecular Biology and Genetics
|
| Lab |
Rachel Green
|
| Street address |
725 N. Wolfe St.
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platforms (1) |
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| Samples (5)
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| Relations |
| BioProject |
PRJNA420568 |
| SRA |
SRP125948 |
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