TABLE 1.
gRNAs and diagnostic IDAA primers for each of the four target regions. Scores and first selection of gRNAs were obtained by feeding ca 1 kb regions to the in silico prediction servers CHOPCHOP (http://chopchop.cbu.uib.no/), CRISPRater (https://crispr.cos.uniheidelberg.de/) and SSC (http://crispr.dfci.harvard.edu/SSC/).
| Diagnostic PCR(s) | gRNA | |
|---|---|---|
| GWD—5′ exon 1 | GWD Forward primer 1 | gJ: TCAGTGGTAAGTACAGCATG |
| 5′ TTTGTATTGACTGATTTTGTATTGT 3′ | gK: AGGGAATAACTTGCTGTACC | |
| GWD Reverse primer 1 | gL: GTTTCGAGGTAACAGGTTAA | |
| FAM 5′ TAGTTTCTAAGCCCCAAGCA3′ | gM: GTACAGCAAGTTATTCCCTA | |
| GWD—3′ exon 24 + 25 | GWD Forward primer 2: | gA: GGAGAGGAGGAAATTCCTGA |
| 5′ TCAGTCCAGTTGAAGCCGTTG 3′ | gB: TGTTCGAGCTAGAAATGGGA | |
| GWD Reverse primer 2: | gC: GCTGACCTCCAAGCAAAGGA | |
| FAM 5′ TCACGAGTTCATTCATCTTTCCCA 3′ | gD: ATTGGCTGACCTCCAAGCAA | |
| gE: TTTCTGTTCGAGCTAGAAAT | ||
| gI: CACAACGACAACATATCCAA | ||
| DMR6—5′ exon 1 | DMR6 Forward primer 1 | g43: TTTGAGGGAGAGTAGAGTGG |
| FAM 5′ CCATGGAAACGAAAGTTATTTC 3′ | g44: GTGGCCTATCGGATTCGGGT | |
| DMR6 Reverse primer primer 1 | ||
| 5′ CAACCTAAGTCAATTATTGGAAC 3′ | ||
| DMR6—5′ exon 2 | DMR6 Forward primer 2 | g45: TGGAGAAATATGCTCCTGAA |
| 5′ AGCTGACCGGCAGCAAAATTGGGTAGCTGGGGAATTTTTCA 3′ | ||
| DMR6 Reverse primer 2 | ||
| 5′ GGTTACCATGCATAACTATACACAC 3′ | ||
| FAM primer | ||
| FAM 5′ AGCTGACCGGCAGCAAAATTG 3′ | ||
| DMR6—5′ exon 1 + 2 | DMR6 Forward primer 1 | g43: TTTGAGGGAGAGTAGAGTGG |
| FAM 5′ CCATGGAAACGAAAGTTATTTC′3 | g44: GTGGCCTATCGGATTCGGGT | |
| DMR6 Reverse primer 2 | g45: TGGAGAAATATGCTCCTGAA | |
| 5′ GGTTACCATGCATAACTATACACAC 3′ | ||
| DMR6 Reverse primer 4 | ||
| FAM 5′ CGATGGATTAGAAGGCCATTC 3′ | ||
| DMR6—3′ exon 3 | DMR6 Forward primer primer 3 | g46: GAAGCCATAGCAGAGAGCCT |
| 5′ ATCGTGAGCAGATATTGCACG 3′ | g47: GAATTTGGATCAGTATGGGC | |
| DMR6 Reverse primer 3 | g48: ATCACCAAGATTAATGACAA | |
| FAM 5′ GGTTTACCTGCAATTGATCAC 3′ |