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Fig. 5 | Plant Methods

Fig. 5

From: Ribozyme-mediated CRISPR/Cas9 gene editing in pyrethrum (Tanacetum cinerariifolium) hairy roots using a RNA polymerase II-dependent promoter

Fig. 5

Ribozyme-based CRISPR/Cas9 vector construction process. The hammerhead-type ribozyme sequence is labeled HH, and the hepatitis delta virus ribozyme sequence is labeled HDV. Step 1: The 229-bp fragment encompassing the ‘SacI + HH + sgRNA + HDV + BamHI’ fragment was obtained. Next, this fragment was digested by SacI and BamHI. Step 2: The 288-bp fragment encompassing the ‘BamHI + NOS + Aaul’ fragment was obtained. Next, this fragment was digested by BamHI. Step 3: The 853-bp fragment encompassing the ‘HindIII + 35S + SacI’ fragment was obtained. Next, this fragment was digested by SacI. Step 4: Their digestion products were mixed at the same molar concentration and ligated. Next, using the ligated DNA mixture as a template, a 1346-bp fragment encompassing the ‘HindIII + 35S + HH + sgRNA + HDV + NOS + Aaul’ was obtained. Step 5: The pRGEB32-GhU6.7-NPT2 vector and the ‘HindIII + 35S + HH + sgRNA + HDV + NOS + Aaul’ fragment were ligated. Next, the plasmid was digested by Aaul. Step 6: The 853 bp fragment ‘Aaul + 35S + Aaul’ was obtained. Next, this fragment was digested by Aaul. Step 7: The digested plasmid in step 5 and the digested fragment ‘Aaul + 35S + Aaul’ were connected

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