CRISPR-mediated genome editing in poplar issued by efficient transformation
CRISPR 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 is often used for replacing nucleotides with one donor DNA template (DDT), including homologous sequences. CRISPR-Cas9 has been recruited, and three variables, Agrobacteria inoculator concentration, the ratio of pDDT/pgRNA, and homologous arm length, have been designed to integrate nptII and 2XCamV35S into the MKK2 promoter zone. Here, we showed that recovered poplars on kanamycin-supplemented media exhibited enhanced expression of MKK2 affected by precise integrated 2XCamV35S 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. Efficient transformations resulted from optimized variables, directly affecting the HDR efficiency through woody plants such as poplar.
Steps to reproduce
Efficient transformation methods have been used to knock in exogenous nptII and promoter 2XCamV35S leading to occur efficient HDR.