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Fig. 1 | Plant Methods

Fig. 1

From: Editing of the urease gene by CRISPR-Cas in the diatom Thalassiosira pseudonana

Fig. 1

Overview of level 1 (L1) and level 2 (L2) Golden Gate cloning for assembly of the CRISPR-Cas construct pAGM4723:TpCC_Urease. L0 modules are created by PCR amplifying the relevant insert, BsaI sites are added through the primers. Products are then TOPO TA cloned into pCR8/GW/TOPO vectors. Construction of the L1 and L2 modules is based on the ability of BsaI and BpiI restriction enzymes to cut outside of their restriction recognition sites leaving 4 nt overhangs specific to each module. Overhangs between adjacent modules correspond allowing multiple modules to be ligated in a particular order within the same reaction. BsaI is used to assemble L0 modules into L1 destination vectors and BpiI is used to assemble L1 modules into the final L2 destination vector. L1 assemblies of pICH47742:FCP:Cas9YFP and pICH47751:U6:sgRNA_Urease 1 are shown. L2 assembly of the final construct is shown. L1 modules containing the FCP:NAT cassette, U6:sgRNA Urease 2 cassette, the L4E linker and L2 destination vector are shown in a simplified format. Corresponding colours denote a shared 4 nt overhang

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