Efficient scar-free knock-ins of several kilobases in plants by engineered CRISPR-Cas endonucleases

• Published in: Molecular plant Vol. 17; no. 5; pp. 824 - 837
• Main Authors: Schreiber, Tom, Prange, Anja, Schäfer, Petra, Iwen, Thomas, Grützner, Ramona, Marillonnet, Sylvestre, Lepage, Aurélie, Javelle, Marie, Paul, Wyatt, Tissier, Alain
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 06.05.2024
• Subjects: 5′ exonuclease; CRISPR-Cas; gene replacement; homology-directed repair; knockin; plants; CRISPR-Cas; 5′ exonuclease; knockin; gene replacement; homology-directed repair; plants
• ISSN: 1674-2052; 1752-9867
• DOI: 10.1016/j.molp.2024.03.013

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. 5′ end resection is a key step in the pathways for homology-directed repair of DNA double-strand breaks. Fusions of CRISPR-Cas9 endonuclease to specific 5′ exonucleases lead to significant increase in scar-free multikilobase knockins in Nicotiana benthamiana, Arabidopsis thaliana, and hexaploidy wheat (Triticum aestivum).