Table 1. Oligonucleotides used in this study.
| Sequence (5ʹ → 3ʹ) | Description |
|---|---|
|
GATTTTCGCTGTCGCACTCTTCATGGGTGCGTGGATTGAAAT
ATTGAcgatagTCAATATTTCAATCCACGCACCCATGAAGAGTGC GACAGCGAAAATC |
CRISPR hairpin target* |
|
GATTTTCGCTGTCGCACTCTTCATGGGTGCGTGGATTGAAAT
ATTGAGGTAGGTATTG |
Mini-CRISPR array |
| CAATACCTACCTCAATATTTCAATCCACGCACCC ATGAAGAGTGCGACAGCGAAAATC |
RC† |
| CGTAGCTGAGGACCACCAGAACAG TTTTGAATTTTTTTT | 15-nt 3ʹ overhang prespacer, 4-nt between duplex and PAM†† |
| CGTAGCTGAGGACCACCAGAACAG TTGAATTTTTTTTTT | 2-nt between duplex and PAM |
| CGTAGCTGAGGACCACCAGAACAG TTTTTTGAATTTTTT | 6-nt between duplex and PAM |
| CGTAGCTGAGGACCACCAGAACAG TTTTTTTTGAATTTT | 8-nt between duplex and PAM |
| CTGTTCTGGTGGTCCTCAGCTACG TTTTGAATTTTTTTT | RC of previous four oligos |
| GATTTTCGCTGTCGCACTCTTCATGGGTGCGTGGATTGAAATATTGA | CRISPR DNA substrate |
| TCAATATTTCAATCCACGCACCCATGAAGAGTGCGACAGCGAAAATC | RC |
| GCGTAGCTGAGGACCACCAGAACAGTTTTGAATTTTTTTTTTTTTTTTTT | 25-nt 3ʹ overhang prespacer |
| GCGTAGCTGAGGACCACCAGAACAG | 25 bp duplex |
| CTGTTCTGGTGGTCCTCAGCTACGC | RC |
| GCGTAGCTGAGGACCTTTTTTTTTTTTTGAATTTTTTTTTTTTTTCAGGT CGACAAGCTTG | T-rich ssDNA prespacer |
| CAAGCTTGTCGACCTGAAAAAAAAAAAAAATTCAAAAAAAAAAAAA GGTCCTCAGCTACGC |
RC |
| CTAGTATGATCATGTCCAACGAATCAATACCTACCTCAATGAACGGAT | 48 bp duplex |
| ATCCGTTCATTGAGGTAGGTATTGATTCGTTGGACATGATCATACTAG | RC |
| GCGTAGCTGAGGACCTTTTTTTTTTTTTTTTTTGAATTGAATTGAA
TTTTTTTTTTTTTTTTTTGACAAGCTTGCGACA |
3 PAM sites interspersed in 2-nt |
| TGTCGCAAGCTTGTCAAAAAAAAAAAAAAAAAATTCAATTCAATTCA AAAAAAAAAAAAAAAAAGGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTTTTTTTTTTTTTTTTTTTTGAAGAAGAATTTTT TTTTTTTTTTTTTTTGACAAGCTTGCGACA |
3 PAM sites without spacing |
| TGTCGCAAGCTTGTCAAAAAAAAAAAAAAAAAAAATTCTTCTTCAAA AAAAAAAAAAAAAAAAAGGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTTTTTTTTTTGAATTTTTTTTTTGAATTTT TTTTTTGAATTTTTTTTTTGACAAGCTTGCGACA | 3 PAM sites interspersed with 10-nt |
| TGTCGCAAGCTTGTCAAAAAAAAAATTCAAAAAAAAAATTCAAAAA AAAAATTCAAAAAAAAAAGGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTTTTTTTTTTTTGAATTTTTTTTGAATTTTTTTT GAATTTTTTTTTTTTGACAAGCTTGCGACA |
3 PAM sites interspersed with 8-nt |
| TGTCGCAAGCTTGTCAAAAAAAAAAAATTCAAAAAAAATTCAAAAAAAA TTCAAAAAAAAAAAAGGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTTTTTTTTTTTTTTGAATTTTTTGAATTTT TTGAATTTTTTTTTTTTTTGACAAGCTTGCGACA |
3 PAM sites interspersed with 6-nt |
| TGTCGCAAGCTTGTCAAAAAAAAAAAAAATTCAAAAAATTCAAAA AATTCAAAAAAAAAAAAAAGGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTTTTTTTTTTTTTTTTGAATTTTGAATTTTGAA
TTTTTTTTTTTTTTTTGACAAGCTTGCGACA |
3 PAM sites interspersed with 4-nt |
| TGTCGCAAGCTTGTCAAAAAAAAAAAAAAAATTCAAAATTCAAAA TTCAAAAAAAAAAAAAAAAGGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTATATATATATATGAATATATATATATATA CAGGTCGACAAGCTTG | AT-rich ssDNA prespacer |
| CAAGCTTGTCGACCTGTATATATATATATATTCATATATATAT ATAGGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTTGGTATTCAACAGAATTTTTTTTTTTTTTCA GGTCGACAAGCTTG |
Non-T-rich upstream/T rich downstream ssDNA prespacer |
| CAAGCTTGTCGACCTGAAAAAAAAAAAAAATTCTGTTGAATACCAAG GTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTTTTTTTTTTTTTGAACTCGTATTCAACAG CAGGTCGACAAGCTTG | T-rich upstream/non T-rich downstream ssDNA prespacer |
| CAAGCTTGTCGACCTGCTGTTGAATACGAGTTCAAAAAAAAAAAAA GGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACCTTGGTATTCAACAGAACTCGTATTC AACAGCAGGTCGACAAGCTTG | Non-T-rich up- and downstream ssDNA prespacer |
| CAAGCTTGTCGACCTGCTGTTGAATACGAGTTCTGTTGAATACCAA GGTCCTCAGCTACGC |
RC |
| GCGTAGCTGAGGACC | Primer used for ddNTP Sanger sequencing |
| GCGTAGCTGAGGACCCGTGGCACCGACATGGCATTTTTNNNNGAA TTTTTGCTGGGCGCTAAGGGACAACTCCAGGTCGACAAGCTTG | NNNN on upstream region |
| GCGTAGCTGAGGACCCGTGGCACCGACATGGCAGTTTTTNNNGAA TTTTTGCTGGGCGCTAAGGGACAACTCCAGGTCGACAAGCTTG | NNN on upstream region |
| GCGTAGCTGAGGACCCGTGGCACCGACATGGCAGGTTTTTNNGAA TTTTTGCTGGGCGCTAAGGGACAACTCCAGGTCGACAAGCTTG | NN on upstream region |
| GCGTAGCTGAGGACCCGTGGCACCGACATGGCAGGCTTTTTNGAA TTTTTGCTGGGCGCTAAGGGACAACTCCAGGTCGACAAGCTTG | N on upstream region |
| GCGTAGCTGAGGACCCGTGGCACCGACATGGCATTTTTGAANNNN TTTTTGCTGGGCGCTAAGGGACAACTCCAGGTCGACAAGCTTG | NNNN on downstream region |
| GCGTAGCTGAGGACCCGTGGCACCGACATGGCATTTTTGAANNNTTTTT CGCTGGGCGCTAAGGGACAACTCCAGGTCGACAAGCTTG | NNN on downstream region |
| GCGTAGCTGAGGACCCGTGGCACCGACATGGCATTTTTGAANN TTTTTCAGCTGGGCGCTAAGGGACAACTCCAGGTCGACAAGCTTG | NN on downstream region |
| GCGTAGCTGAGGACCCGTGGCACCGACATGGCATTTTTGAAN TTTTTCATGCTGGGCGCTAAGGGACAACTCCAGGTCGACAAGCTTG |
N on downstream region |
| CAAGCTTGTCGACCTG | Primer used for primer extension |
| TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAAGCTTGTCGACCTG | Primer used for amplification-Forward |
| GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGCGTAGCTGAGGACC | Primer used for amplification-Reverse |
*For CRISPR oligonucleotides, leader is in italics, repeat is in bold, and spacer is in plain uppercase font. For hairpin, the loop region is in lowercase.
†RC = reverse complement of previous oligonucleotide.
††For cleavage substrates, PAM sequences are underlined.