Table 2. Details of primers used in the study.
| Purpose | Primer combinations and sequence (5’-3’) | Amplification condition* | Expected amplicon size (kb) | |
|---|---|---|---|---|
| Annealing temperature (0C) | Extension time (Sec) at 72°C | |||
| Amplification of all the target site for in vitro cleavage assay | ChiLCV-C1/V1q F (GGGCTAAGGTCGAGATGTCC)ChiLCV-C1/V1 R (CATCCATCCATATCTTCCCTAATAC) | 59 | 45 | 1.5 |
| Generating amplicon for T7E1 assay and sequencing | ChiLCV-C1/V1q F (GGGCTAAGGTCGAGATGTCC)ChiLCV IR-R (GTCGCTTGGACATAATTCTTAGC) | 61.2 | 37 |
1.2 |
|
ChiLCV-IR F (ATTGTATGAGGACGTGGAGATGAG) ChiLCV-C1/V1 R (CATCCATCCATATCTTCCCTAATAC) |
60.7 | 30 | 1.0 | |
| qPCR of virus quantification | ChiLCV-C1/V1q F (GGGCTAAGGTCGAGATGTCC)ChiLCV-q R (CCGGAGGAACTTGAAGAATGG) | 62 |
30 | 0.2 |
| qPCR of gRNA expression | ||||
| i. gRNA-1 | IR-spacer3-Top (GGTCAGCCATCCGCACTAATATTAC)gR-qR (GCACCGACTCGGTGCCAC) | 64 | 30 | 0.096 |
| ii. gRNA-2 | IR-spacer6-Top (GATTGCATGGTCCCCCCTATAAACT)gR-qR: (GCACCGACTCGGTGCCAC) | 64 | 30 | 0.096 |
| iii. gRNA-3 | V2/V1-Spacer1-Top (GGTCACATTTCCACGCCCGCCTCGA)gR-qR: (GCACCGACTCGGTGCCAC) | 64 | 30 | 0.096 |
| iv. gRNA-4 | V2/V1-Spacer3-Top (GATTGAGGCCAGAGCATGGGTGAAC)gR-qR: (GCACCGACTCGGTGCCAC) | 64 | 30 | 0.096 |
| v. gRNA-5 | C1/C4-spacer1-Top (GGTCAGGACCCTGAATTGATTGCCT)gR-qR: (GCACCGACTCGGTGCCAC) | 64 | 30 | 0.25 |
| vi. gRNA-6 | C1/C4-spacer2-Top (GATTGTAGCTGATCTTCCATCGACT)gR-qR: (GCACCGACTCGGTGCCAC) | 64 | 30 | 0.50 |
| qPCR for Cas9 | Cas9 qF (GGACCACTTGCTAGAGGAAACTCTC) Cas9 qR (GGAAGGTTCTTATCGAAGTTGGTCATTC) | 64 | 30 | 0.15 |
| PP2A gene primer of N. benthamiana(reference gene control on qPCR assay for gRNA and Cas9) | PP2A F (GACCCTGATGTTGATGTTCGCT)PP2A R (GAGGGATTTGAAGAGAGATTTC) | 59 | 30 | 0.2 |
| Actin gene primer of N. benthamiana(reference gene control on qPCR assay for ChiLCV) |
Actin-DNA-qF(AATGATCGGAATGGAAGCTG) Actin-DNA-qR: (TGGTACCACCACTGAGGACA) |
61.2 | 30 | 0.116 |
*other parameters of PCR are followed from standard methods. Phusion Taq polymerase (Thermo) was used.