Stbl3 competent e coli cells

Genomic DNA was isolated from single cell clones using Quick Extract-DNA extraction solution according to the manufacturer’s instructions (Epicenter). The extracted DNA served as a template to amplify the Prnp gene with primers PrP-F/PrP-R (Supplementary Table 1) using Q5 DNA polymerase. The PCR conditions were 94 °C for 5 min, followed by 40 cycles of 94 °C for 45 s, 59 °C for 45 s, 72 °C for 60 s, and 7 min at 72 °C. The PCR products were purified using QIAquick PCR purification kit, cloned into pCR–blunt TOPO cloning kit. The ligated mixture was transformed into Stbl3 competent E. coli cells (Thermo Fisher Scientific). Plasmid DNA was isolated from twelve of the bacterial colonies per cell clone and sent for Sanger sequencing using the PrP-F and PrP-R primers.

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).
Table 2

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)]