Table 3. Sequence of double stranded spacer oligos along with linker and the corresponding Golden Gate entry vectors where they were cloned to generate gRNAs.
| Selected Spacers | Two strands of spacer oligo sequence with linker (5'-3')* | Entry vector (promoter) | Name of gRNA Constructs |
|---|---|---|---|
| IR-Spacer3 | IR-spacer3-Top:ggtcaGCCATCCGCACTAATATTAC | pYPQ131B (U3) | gRNA1 |
| IR-spacer3-Bot:aaacGTAATATTAGTGCGGATGGCt | |||
| IR-Spacer6 | IR-spacer6-Top: gattgCATGGTCCCCCCTATAAACT | pYPQ132A (U6) | gRNA2 |
| IR-spacer6-Bot: aaacAGTTTATAGGGGGGACCATGc | |||
| V2/V1-Spacer1 | V2/V1-Spacer1-Top: ggtcaCATTTCCACGCCCGCCTCGA | pYPQ131B (U3) | gRNA3 |
| V2/V1-Spacer1-Bot: aaacTCGAGGCGGGCGTGGAAATGt | |||
| V2/V1-Spacer3 | V2/V1-Spacer3-Top: gattgAGGCCAGAGCATGGGTGAAC | pYPQ132A (U6) | gRNA4 |
| V2/V1-Spacer3-Bot: aaacGTTCACCCATGCTCTGGCCTc | |||
| C1/C4-Spacer1 | C1/C4-spacer1-Top: ggtcaGGACCCTGAATTGATTGCCT | pYPQ131B (U3) and pYPQ133B (U3) | gRNA5 gRNA5-1 |
| C1/C4-spacer1-Bot: aaacAGGCAATCAATTCAGGGTCCt | |||
| C1/C4-Spacer2 | C1/C4-spacer2-Top: gattgTAGCTGATCTTCCATCGACT | pYPQ132A (U6) | gRNA6 |
| C1/C4-spacer2-Bot: aaacAGTCGATGGAAGATCAGCTAc |
*Spacer sequences (Top) and its complimentary sequences (Bot) were synthesized with addition of linker (underlined) as required for their cloning into GoldenGate entry vectors. The Top and Bot sequences were annealed to each other to develop double stranded spacer molecule for cloning. The vector has the promoter and gRNA scaffold. After cloning the spacer sequence along with scaffold form the functional gRNAs.