Site‐directed integration of exogenous DNA into the soybean genome by LbCas12a fused to a plant viral HUH endonuclease.

Autor: Nagy, Ervin D., Kuehn, Rosemarie, Wang, Dafu, Shrawat, Ashok, Duda, David M., Groat, Jeanna R., Yang, Peizhen, Beach, Steven, Zhang, Yuanji, Rymarquis, Linda, Carter, Sharina L., Gaeta, Robert T., Gilbertson, Larry A.
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Zdroj: Plant Journal; Aug2022, Vol. 111 Issue 3, p905-916, 12p
Abstrakt: SUMMARY: High efficiency site‐directed chromosomal integration of exogenous DNA in plants remains a challenge despite recent advances in genome editing technologies. One approach to mitigate this problem is to increase the effective concentration of the donor DNA at the target site of interest. HUH endonucleases (ENs) coordinate rolling circle replication. In vitro, they can form stable covalent bonds with DNA that carries their recognition motifs. When fused to a CRISPR‐associated endonuclease, HUH ENs may improve integration rates by increasing the local donor concentration through tethering of the donor to the CRISPR nuclease. We tested this hypothesis by using chimeric proteins between LbCas12a as a CRISPR‐associated endonuclease and the HUH EN from Faba Bean Necrotic Yellow Virus in soybean (Glycine max). Two fusion protein configurations were tested to integrate a 70‐nt oligonucleotide donor into a commercially important target site using protoplasts and in planta transformation. Site‐directed integration rates of the donor DNA, when tethered to the fusion protein, reached about 26% in plants and were up to four‐fold higher than in untethered controls. Integrations via canonical homology‐directed repair or non‐homologous end joining were promoted by tethering in a similar fashion. This study is the first demonstration of HUH EN‐associated tethering to improve site‐directed DNA integration in plants. Significance Statement: An exceptionally high rate of site‐directed integration of exogenous DNA into the soybean (Glycine max) genome was achieved using a novel method to increase the local donor DNA concentration around the CRISPR target site of interest. A donor DNA was covalently tethered to a fusion protein between LbCas12a and the HUH endonuclease of Faba Bean Necrotic Yellow Virus, which was delivered into soybean protoplasts and plants as part of a ribonucleoprotein complex. [ABSTRACT FROM AUTHOR]
Databáze: Complementary Index
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