Table_4_CRISPR-mediated genome editing in poplar issued by efficient transformation.xlsx

BackgroundCRISPR has been increasingly used for plant genetic improvements because of its high efficiency and precision. Recently, the authors have reported the possibility of homology-directed repair (HDR) using CRISPR/Cas9 through woody plants such as poplar. HDR often replaces nucleotides with one donor DNA template (DDT), including homologous sequences.MethodsCRISPR–Cas9 was recruited, and three variables, Agrobacteria inoculator concentration, pDDT/pgRNA ratio, and homologous arm length, were designed to integrate nptII and 2XCamV 35S into the MKK2 promoter zone.ResultsHere, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by the precise integration of 2XcamV 35S and nptII, improving biochemical and phenotypic properties. Our findings confirmed that Agrobacterium inoculator OD600 = 2.5, increased DDT numbers during cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused efficient HDR and increased MKK2 expression.ConclusionEfficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar.

背景：CRISPR技术因其高效性与精确性，在植物遗传改良领域得到了日益广泛的应用。近期，研究者们报道了通过诸如杨树等木质植物利用CRISPR/Cas9进行同源定向修复（HDR）的可能性。HDR通常通过一供体DNA模板（DDT）替换核苷酸，包括同源序列。方法：本研究中，CRISPR–Cas9技术被采用，并设计了三个变量，即农杆菌接种浓度、pDDT/pgRNA比例和同源臂长度，以将nptII和2XCamV 35S整合到MKK2启动子区域。结果：本研究结果表明，在卡那霉素补充培养基上恢复的杨树展现了MKK2表达增强的现象，这是由于2XcamV 35S和nptII的精确整合所导致的，从而改善了生物化学和表型特性。我们的研究证实，农杆菌接种浓度OD600=2.5，在细胞分裂期间将DDT数量增加到4:1的pDDT/pgRNA比例，并优化同源臂长度为700 bp，均能有效地促进HDR过程并增加MKK2的表达。结论：通过优化变量，实现了高效的转化，直接影响了通过杨树等木质植物进行的HDR效率。