Fig. 1
From: Beyond Seek and Destroy: how to Generate Allelic Series Using Genome Editing Tools
Binary plasmids used. From top to bottom. Binary plasmid containing the LbCPF1 sequence (Tang et al. 2017) codon-optimized for rice, under the control of the promoter pZmUBI; crRNA-CAO1 was also under the control of a pZmUBI promoter. The plant resistance marker was hygromycin. Binary plasmid containing SpCAS9 (Miao et al. 2013) codon-optimized for rice under the control of the pZmUBI promoter. sgRNA-CAO1 was under the control of the rice promoter pOsU3. The plant resistance marker was hygromycin. Plasmid containing BEnCAS9 (Zong et al. 2017) codon-optimized for rice (available on Addgene: #98163), formed by a fusion of the rat rAPOBEC1 protein (Komor et al.), the XTEN linker, the nCAS9 nickase having a mutation inactivating the catalytic domain RuvC (D10A) and the UGI protein. The original cloning sites were replaced by AttR Gateway recombination sites. sgRNA-BECAO1 targeting exon 3 of the OsCAO1 gene was under the control of the rice pOsU3 promoter. HDV: HDV ribozyme; HH: Hammerhead ribozyme. All spacers were first cloned into entry vectors and then transferred to the binary vectors by LR reactions