Table 3.
Primers used to construct and verify insertion of CRISPR-Cas9 gRNA in pMEL and pROS plasmid series.
| Name | Sequence 5΄ → 3΄ | Purpose |
|---|---|---|
| 6005 | GATCATTTATCTTTCACTGCGGAGAAG | pMEL + pROS plasmid construction |
| 6006 | GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC | pMEL plasmid construction |
| 2873 | TCAGACTTCTTAACTCCTGTAAAAACAAAAAAAAAAAAAGGCATAGCAATAAGCTGGAGCTCATAGCTTC | Construction Spcas9 integration cassette |
| 4653 | GTGCCTATTGATGATCTGGCGGAATGTCTGCCGTGCCATAGCCATGCCTTCACATATAGTCCGCAAATTA AAGCCTTCGAG | Construction Spcas9 integration cassette |
| 3093 | ACTATATGTGAAGGCATGGCTATGGCACGGCAGACATTCCGCCAGATCATCAATAGGCACCTTCGTACGC TGCAGGTCGAC | Construction of natNT2 integration cassette |
| 5542 | CTATGCTACAACATTCCAAAATTTGTCCCAAAAAGTCTTTGGTTCATGATCTTCCCATACGCATAGGCCA CTAGTGGATCTG | Construction of natNT2 integration cassette |
| Primer α | CACCTTTCGAGAGGACGATG | Confirmation pROS plasmids |
| Primer β | GCTGGCCTTTTGCTCACATG | Confirmation pROS plasmids |
| Ptarget FWa | TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCN 20 GTTTTAGAGCTAGAAATAGC AAGTTAAAATAAGGCTAGTCCGTTATCAAC | pMEL construction |
| Ptarget RVb | GTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACN 20c GATCATTTATCTTTCACTG CGGAGAAGTTTCGAACGCCGAAACATGCGCA | pMEL construction |
| Ptarget ROS | TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCN 20 GTTTTAGAGCTAGAAATAGC AAGTTAAAATAAG | pROS construction |
| Ptargetcas9 ROSc | TGCGCATGTTTCGGCGTTCGAAACTTCTCCGCAGTGAAAGATAAATGATCCTAGGCTGTCCAAATCCCGGGTT TTAGAGCTAGAAATAGCAAGTTAAAATAAG | Construction of a pROS targeting cas9 |
| PrepairD FW | TTTCAGAGTTCTTCAGACTTCTTAACTCCTGTAAAAACAAAAAAAAAAAAAGGCATAGCATATGAGGGTGAGA ATGCGAAATGGCGTGGAAATGTGATCAAAGGTAATAAAACGTCATAT | Repair DNA for cas9 deletion leaving a can1Δ locus |
| PrepairD RV | ATATGACGTTTTATTACCTTTGATCACATTTCCACGCCATTTCGCATTCTCACCCTCATATGCTATGCCTTTT TTTTTTTTTGTTTTTACAGGAGTTAAGAAGTCTGAAGAACTCTGAAA | Repair DNA for cas9 deletion leaving a can1Δ locus |
| PrepairCAN1 FWd | TTTCAGAGTTCTTCAGACTTCTTAACTCCTGTAAAAACAAAAAAAAAAAAAGGCATAGCAATGACAAATTCAA AAGAAGACGCC | Repair DNA for cas9 deletion restoring a CAN1 locus |
| PrepairCAN1 RVd | ATATGACGTTTTATTACCTTTGATCACATTTCCACGCCATTTCGCATTCTCACCCTCATATCTATGCTACAAC ATTCCAAAATTTG | Repair DNA for cas9 deletion restoring a CAN1 locus |
a
N20 corresponds to the chosen target sequence. This nucleotide sequence does not include the PAM sequence (NGG).
b
N20c corresponds to the reverse complement of the chosen target sequence found in Ptarget FW.
c
Primer for the construction of pROS plasmid carrying a spacer targeting cas9, which can be used to remove chromosomal integration of the endonuclease expression cassette.
d
Primer suitable to PCR amplify the CAN1 ORF.