Bayer Crop Science, St. Louis, Missouri, USA.
Plant J. 2022 Aug;111(3):905-916. doi: 10.1111/tpj.15849. Epub 2022 Jun 19.
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.
尽管基因组编辑技术最近取得了进展，但高效的靶向染色体中外源 DNA 整合仍然是一个挑战。一种解决此问题的方法是增加目标位点处供体 DNA 的有效浓度。HUH 内切酶（ENs）协调滚环复制。在体外，它们可以与携带其识别基序的 DNA 形成稳定的共价键。当与 CRISPR 相关的内切酶融合时，HUH EN 可以通过将供体与 CRISPR 核酸酶连接来增加局部供体浓度，从而提高整合率。我们通过使用 LbCas12a 作为 CRISPR 相关内切酶和来自蚕豆坏死黄病毒的 HUH EN 之间的嵌合蛋白在大豆（Glycine max）中测试了这一假设。为了使用原生质体和体内转化将 70nt 寡核苷酸供体整合到商业上重要的靶位点，测试了两种融合蛋白构型。当与融合蛋白连接时，供体 DNA 的靶向整合率在植物中达到约 26%，比未连接的对照高出四倍。通过经典同源定向修复或非同源末端连接的整合也以类似的方式通过连接得到促进。这项研究首次证明了 HUH EN 相关的连接可以提高植物中靶向 DNA 整合的效率。
