Extended Data Table 2. DNA and RNA sequences used in the study.
Please refer to web version for coloured text.
| Description | Sequence* | Used in |
|---|---|---|
| Forward primer for PCR amplification of ssDNA templates, T7 promoter † | 5′–TAATACGACTCACTATA–3′ | in vitro transcription of sgRNA |
| ssDNA template sgRNA 1 † | 5′–CACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC TTTTTTACAAATTGAGTTATCCTATAGTGAGTCGTATTA–3′ |
in vitro transcription of sgRNA 1 |
| sgRNA 1 | 5′–GGAUAACUCAAUUUGUAAAAAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG CUAGUCCGUUAUCAACUUGAAAAAGUG–3′ |
Fig. 1, 2a-c, , 3c,e, ED Fig. 1, 2a,c,d, 6b (crystallography) Fig. 2d,e, ED Fig. 3, 7 |
| Reverse primer for PCR amplification of sgRNA1 ssDNA template | 5′–CACTTTTTCAAGTTGA–3′ | in vitro transcription of sgRNA 1 |
| target strand 1 | 5′–CAATACCATTTTTTACAAATTGAGTTAT–3′ | Fig. 1, 2a-c, 3a-c, ED Fig. 1, 2a,c,d, 6 (crystallography) Fig. 2d |
| target strand 1 ATTO532-5′ | ATTO532–5′-GCGCAATACCATTTTTTACAAATTGAGTTAT–3′ | ED Fig. 3b, 7a,d |
| target strand 2 MM FL ATTO532-5′‡ | ATTO532–5′–GCGCAATACCAAATTTTACAAATTGAGTTAT–3′ | ED Fig. 7b,c,d |
| target strand 2 MM | 5′–CAATACCACAT–3′ | Fig. 3e (crystallography) |
| target strand 3 MM | 5′–CAATACCACAA–3′ | Fig. 3e (crystallography) |
| target strand 1 distal | 5′–TTTACAAATTGAGTTAT–3′ | Fig. 3e (crystallography) |
| non-target strand 1 | 5′–ATAACTCAATTTGTAAAAAATGGTATTG–3′ | ED Fig. 3b, 7a,c,d |
| non-target strand 1 3‘-ATTO532 | 5′–ATAACTCAATTTGTAAAAAATGGTATTGCGC-3′–ATTO532 | Fig. 2d |
| non-target strand 1 2 MM ‡ | 5′–ATAACTCAATTTGTAAAATTTGGTATTG–3′ | ED Fig. 7b,d |
| non-target strand 1 (product) | 5′–AAAATGGTATTG–3′ | Fig. 1, 2a-c, 3a,c, ED Fig. 1, 2a,c,d, 6a (crystallography) |
| non-target strand 2 MM | 5′–ATGTGGTATTG–3′ | Fig. 3e (crystallography) |
| non-target strand 3 MM | 5′–TTGTGGTATTG–3′ | Fig 3e (crystallography) |
| pMJ879 § (target plasmid 1) | 5′–….GAATTCCTAATGCGCCATTTTTTACAAATTGAGTTATGGATCC….-3′ 3′–….CTTAAGGATTACGCGGTAAAAAATGTTTAACTCAATACCTAGG….-5′ |
Fig. 2e, 3d, ED Fig. 3a |
| pMJ891 § (target plasmid 1, 2 MM) | 5′–….GAATTCCTAATGCGCCAAATTTTACAAATTGAGTTATGGATCC….-3′ 3′–….CTTAAGGATTACGCGGTTTAAAATGTTTAACTCAATACCTAGG–5′ |
Fig. 3d |
| ssDNA template sgRNA 2 † | 5′–CACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC GCGTCTCATCTTTATGCGTCCCTATAGTGAGTCGTATTA–3′ |
in vitro transcription of sgRNA 2 |
| sgRNA 2 | 5′–GGGACGCAUAAAGAUGAGACGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGC UAGUCCGUUAUCAACUUGAAAAAGUG–3′ |
ED Fig 5 |
| pMJ992 § (target plasmid 2, NGG PAM) | 5′–…. GAATTCGTGAGACCAGCGTCTCATCTTTATGCGTCGGATCC–3′ 3′–….CTTAAGCACTCTGGTCGCAGAGTAGAAATACGCAGCCTAGG….-5′ |
ED Fig. 5a |
| pMJ993 § (target plasmid 2, NAA PAM) | 5′–…. GAATTCGTGAGATTAGCGTCTCATCTTTATGCGTCGGATCC–3′ 3′–….CTTAAGCACTCTAATCGCAGAGTAGAAATACGCAGCCTAGG….–5′ |
ED Fig. 5b |
| pMJ994 § (target plasmid 2, NAAAA PAM) | 5′–…. GAATTCGTGATTTTAGCGTCTCATCTTTATGCGTCGGATCC–3′ 3′–….CTTAAGCACTAAAATCGCAGAGTAGAAATACGCAGCCTAGG….–5′ |
ED Fig. 5c |
*
sgRNA guide sequences and complementary DNA target strand sequences are shown in red. Non-target strand PAM sites (5′-NGG-3′, 5′-NAA-3′, 5′-NAAA-3′) are highlighted in bold italics. Target-strand complement of the PAM (5′-CCN-3′, 5′-TTN-3′, 5′-TTTTN-3′) is denoted in italic.
†
The sequence corresponding to the T7 promoter is coloured blue.
‡
Nucleotide positions with a mismatch (MM) to the sgRNA guide sequence are underlined.
§
All inserts shown are ligated into pUC19 vector between EcoRI and BamHI restriction (shown in grey).