Table 1.
Primer sequences for sgRNA cloning.
| Name | Sequence | Purpose |
|---|---|---|
| Ec-F | 5′-N(20~25)GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC-3′ | Introduce new base-pairing sequences into E. coli sgRNAs |
| Ec-R | 5′-ACTAGTATTATACCTAGGACTGAGCTAGC-3′ | |
| Ec-F-control | 5′-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGC-3′ | Construct the negative control for the sgRNA vector = without the base-pairing region (PCR with Ec-R) |
| Ec-F-colony | 5′-GGGTTATTGTCTCATGAGCGGATACATATTTG-3′ | Colony PCR primers for E. coli sgRNA vector verification |
| Ec-R-colony | 5′-CGCGGCCTTTTTACGGTTC-3′ | |
| 293T-F | 5′-GGAGAACCACCTTGTTGGN(20~25)GTTTTA GAGCTA GAAATA GCAA GTTAAAATAAGGC-3′ | Construct sgRNAs on U6 vectors. The restriction sites are underlined, and the complementary sequence is in bold |
| 293T-R | 5′-CCTAGTACTCGAGAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGA CTAGCCTTATTTTAACTT GCTATTTCTA GCTCTAAAAC-3′ |
|
| 293T-F-double | 5′-GCCGCGAGATCTGAGATCCGACGCGCCATC-3′ | Construct tandem sgRNAs on the U6 vector |
| 293T-R-double | 5′-AATAACTAATGCATGGCGGTAATACGGT-3′ | |
| 293T-F-seq | 5′-GAGATCCAGTTTGGTTAGTACCGGG-3′ | Sequencing primers for U6 vector |
| 293T-R-seq | 5′-ATGCATGGCGGTAATACGGTTAT-3′ |