Effective transformations result in an efficient HDR DNA repair system in poplar

Genetic improvements and RNA-guided genome editing of plants use clustered regularly interspaced short palindromic repeats (CRISPR) associated with a potential nuclear-localized endonuclease (Cas9). DNA system repairs include homology-directed repair (HDR) and non-homologous end-joining (NHEJ). HDR is often used for replacing nucleotides with one donor DNA template (DDT), including homologous sequences, while NHEJ repairs damaged DNA by inserting or deleting (Indel) nucleotides in DSB regions. We recruited CRISPR-Cas9 and designed three variables, Agrobacteria inoculator concentration, the ratio of pDDT/pgRNA, and homologous arm length, to integrate nptII and 2XCamV35S into the MKK2 promoter zone. Here, we showed that recovered events on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by precise integrated 2XCamV35S and nptII, improving biochemical and phenotypic properties. Our data verified efficient transformation achieved by Agrobacterium inoculator OD600=2.5, increased DDTs numbers among cell division to 4:1 pDDT/pgRNA, and optimized homologous arms 700 bp caused to occur efficient HDR leading to more MKK2 expression.

植物的遗传改良与RNA靶向基因组编辑技术，通常借助成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats, CRISPR）结合潜在核定位核酸内切酶Cas9实现。DNA修复系统包含同源定向修复（homology-directed repair, HDR）与非同源末端连接（non-homologous end-joining, NHEJ）：同源定向修复通常借助单条供体DNA模板（donor DNA template, DDT）完成核苷酸替换，模板包含同源序列；而非同源末端连接则通过在双链断裂（double-strand break, DSB）区域插入或缺失核苷酸（insertions/deletions, Indel）来修复受损DNA。本研究采用CRISPR-Cas9系统，设置农杆菌接种浓度、pDDT/pgRNA比值及同源臂长度三个变量，将nptII与2XCamV35S整合至MKK2启动子区域。研究发现，在添加卡那霉素的培养基上获得的再生株系，因精准整合了2XCamV35S与nptII，其MKK2基因表达量显著上调，同时优化了植株的生化与表型特性。本研究数据证实：当农杆菌接种浓度的光密度值OD600为2.5时，转化效率最优；在细胞分裂阶段将pDDT/pgRNA比值提升至4:1，可增加供体DNA模板的有效数量；将同源臂优化至700 bp时，可诱导高效的同源定向修复，进一步提高MKK2基因的表达水平。