Part:BBa_K3001021
Lwa crRNA targeting RNA Mango with T7 promoter
This CRISPR RNA (crRNA) is designed to interact with Cas13a isolated from Leptotrichia wadei (Lwa). It is designed to target snR30-RNA Mango II RNA transcript. It is designed to be in vitro transcribed using T7 polymerase. This construct was synthesized in pUC19.
CRISPR RNA (crRNA) is a crucial aspect of the CRISPR Cas13a system. It seeks out the target RNA sequence so that the Cas13a can cleave the RNA. Our crRNA sequences were synthesized by IDT into the plasmid pUC19. Then we successfully PCR amplified the DNA out of the pUC19 plasmid.
Figure 1. 10% DNA PAGE of crRNA products for Lsh and Lwa. Left to right: lane 1: 100 bp ladder (Thermo Scientific. Lane 2: Lwa 56.5; Lane 3: Lwa 56.8; lane 4: Lsh 56.5; lane 5: Lsh 56.8. Note that all lanes are from the same gel.
Figure 2. 10% DNA PAGE of Lbu and Lwa PCR products. Left to right: lanes 1-6: Lbu; lanes 7-12: Lwa; lane 13: 100 bp ladder; lanes 14-15: empty.
Due to time constraints, our team chose to complete a large scale in vitro transcription using only the Lwa and Lbu crRNAs (Figure 3). This allowed our team to use the crRNA with our other project components in later experiments.
Figure 3. Urea PAGE of in vitro transcribed Lbu and Lwa crRNAs. Left to right: lane 1: High Range RiboRuler; lane 2: Lwa after DNase; lane 3: Lwa after in vitro transcription; lane 4: Lwa before in vitro transcription; lane 5: Lbu after DNase; lane 6: Lbu after in vitro transcription; lane 7: Lbu before in vitro transcription. Note that all lanes are from the same gel.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 71
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 71
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 71
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 71
- 1000COMPATIBLE WITH RFC[1000]
| None |