| RRC ID |
85158
|
| Author |
Schreiber T, Prange A, Schäfer P, Iwen T, Grützner R, Marillonnet S, Lepage A, Javelle M, Paul W, Tissier A.
|
| Title |
Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases.
|
| Journal |
Mol Plant
|
| Abstract |
In plants and mammals, non-homologous end-joining is the dominant pathway to repair DNA double-strand breaks, making it challenging to generate knock-in events. In this study, we identified two groups of exonucleases from the herpes virus and the bacteriophage T7 families that conferred an up to 38-fold increase in homology-directed repair frequencies when fused to Cas9/Cas12a in a tobacco mosaic virus-based transient assay in Nicotiana benthamiana. We achieved precise and scar-free insertion of several kilobases of DNA both in transient and stable transformation systems. In Arabidopsis thaliana, fusion of Cas9 to a herpes virus family exonuclease led to 10-fold higher frequencies of knock-ins in the first generation of transformants. In addition, we demonstrated stable and heritable knock-ins in wheat in 1% of the primary transformants. Taken together, our results open perspectives for the routine production of heritable knock-in and gene replacement events in plants.
|
| Volume |
17(5)
|
| Pages |
824-837
|
| Published |
2024-5-6
|
| DOI |
10.1016/j.molp.2024.03.013
|
| PII |
S1674-2052(24)00086-8
|
| PMID |
38520090
|
| MeSH |
Arabidopsis / enzymology
Arabidopsis / genetics
CRISPR-Cas Systems* / genetics
Endonucleases / genetics
Endonucleases / metabolism
Gene Knock-In Techniques*
Nicotiana* / genetics
Plants, Genetically Modified
Triticum / genetics
|
| IF |
12.084
|
| Resource |
| Wheat |
LPGKU2304 |
