pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W (pKLV2) was a gift from Kosuke Yusa (Addgene plasmid # 67974). Specific gRNA for EGFP and RIG-I (Table 2 , underlined) were cloned according to Golden Gate reaction from ZhangLab protocol (Addgene SAM library sgRNA cloning protocol) using BbsI-HF (NEB) and Stbl3 competent E. coli cells (Thermo Fisher) for transformation. Briefly, 1 μL of each oligo (100 μM) were mixed with 1 μL of T4 ligation buffer (NEB) and 7 μL of water. The mix was heated to 95 °C for 5 min and cooled to room temperature. 1 μL of the annealed oligos was mixed with 25 ng of pKLV2, 1 μL of T4 ligation buffer, 9 μL of water and 0.5 μL of BbsI enzyme and 0.5 μL of T4 ligase enzyme (200 U, NEB). The mix was incubated for 10 cycles of 5 min at 37 °C and 5 min at 23 °C. Five μL were transformed in 50 μL competent E. coli (Stbl3, Thermo fisher). Four colonies were picked and grown in LB + Carbenicillin, the plasmids purified (NEB Monarch Plasmid miniprep) and sequenced to verify correct insertion using U6_Fw_seq primer (Table 1 ).
Plasmids used in this study (from Addgene, USA)
| Plasmid construct | Plasmid name (Addgene) | Plasmid number (Addgene) |
|---|---|---|
| pLenti CMV:EGFP_PGK:Puro | pLenti CMV GFP Puro (658–5) | 17,448, [43 (link)] |
| pLenti hU6:gRNA_PKG:Puro | pKLV2-U6gRNA5(BbsI)-PGKpuro2ABFP-W | 67,974, [21 (link)] |
Free full text: Click here
