Sgrna

CRISPR interference-mediated inhibition of pir (orf23) expression was performed as described previously (40 (link)) using pdCas9 (Addgene, USA; catalogue number 44249) and sgRNA (Addgene, USA; catalogue number 44251) plasmids (41 (link)). The guide RNA (gRNA) plasmid was customized by site-directed mutagenesis by the use of forward primer EcF_LS2, carrying the 20-nucleotide (nt) base-pairing sequence (5′-TAGAACCCGCAACGCTGGCG-3′), and reverse primer EcR (see Table S1). The base pairing region was designed to target the pir gene on T1 phage DNA on the coding strand adjacent to protospacer motif AGG. dCas9 protein expression was induced with 5 ng/ml aTc in both overnight and day cultures.

Lentiviral transduction: HEK-293T were grown to 60–70% confluence in a 60 mm plate, then transfected with sgRNA (CRISPR/Cas9 system lentiCRISPRv2.0 backbone, plasmid 52961) (Addgene, Watertown, MA, USA) or lentiviral expression vectors (LV-h-SCAMP3 ORF) (Vector Builder, Chicago, IL, USA) together with packaging plasmids (PCMV delta R8.2 and pMD2. G (Adggene, Watertown, MA, USA). FuGENE HD (Promega) was used as the transfection reagent according to the manufacturer’s instructions. After 18 h posttransfection, cells were refreshed with DMEM supplemented with 30% FBS for viral particle stability and the supernatant was collected at 48 h. The cells were refreshed again, and the supernatant was collected the next day. Finally, the particles were concentrated using the Lenti-X™ Concentrator (Takara, Kusatsu, Shiga, Japan) for 72 h and stored at −80 °C. Cancer cells were transduced with the virus and selected in puromycin.
siRNA transfections: MDA-MB-231 cells were seeded in a six-well plate until 70% confluent. Cells were transfected with SCAMP3 targeting siRNA and nontargeting sequences (control) at a final concentration of 50 nM using FuGENE HD^® (Promega, Madison, WI, USA). SCAMP3 siRNA (sc-41294) and scrambled siRNA (sc-37007) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

NMuMG cells were plated at 10⁵ cells/well in a six-well plate 24 h before transfection. Cells were transfected with targeting vector (LITMUS29 backbone), sgRNA (Addgene plasmid #41824), and Cas9 expression vector (pCMVsp6-nls-hCas9-nls), a gift from Li-En Jao (UC Davis), at an equimolar (550 fmol) ratio using Xfect transfection reagent according to the manufacturer’s protocol. The GFP-positive cells were subsequently sorted on a FACSAriaIII (BD) 5 d post transfection. After single-cell cloning, insertions were confirmed by immunoblot analysis and PCR-based genotyping. Sequences of sgRNA sequences and primers are available in Supplementary Table 2.

In order to activate endogenous CDK13 we used a CRISPR-Cas9 complex with three SAM components: dCas9–VP64 (Addgene #61425),MS2–p65–HSF1(Addgene, #61426), and sgRNA (Addgene, #89493) as described previously [31 (link)]. gRNAs were designed using the online optimized CRISPR design tool (http://crispr.mit.edu) and targeted the proximal promoter regions of CDK13. Oligos, synthesized by Sangon Biotech., (Shanghai, China), were annealed and sub-cloned into the lentiGuide-puro vector.