Gibson assembly master mix

The shP3F vector was made by cloning the shP3F sequence into the pCDH-LMN vector with a T2A-GFP expression cassette (45 (link)).
The dCas9-KRAB-BFP, mU6 sgRNA-Puro-T2A-GFP, mU6 sgRNA-Puro-T2A-BFP, and hU6 sgRNA-Puro-T2A-BFP plasmids were gifts from Dr. Jonathan Weissman (Whitehead Institute). To generate dual sgRNA constructs, the mU6 sgRNA-Puro-T2A GFP vector was digested with XbaI and KpnI, and the 3.9 kb fragment was ligated into the 5.5 kb backbone of the hU6 sgRNA-Puro-T2A GFP vector digested with AvrII and KpnI.
To generate dual sgRNA and shRNA constructs, the shP3F construct was cloned into the pLKO vector (Addgene). The mU6 sgRNA-Puro-T2A-GFP or -BFP cassette was then digested with XbaI and KpnI, and the 3.9 kb fragment was ligated into the 6.1 kb backbone of pLKO-shP3F digested with SpeI and KpnI.
WT or mutant cDNAs of NRAS were synthesized as gBlocks (IDT), then cloned into pLV1-Hygro with a Gibson Assembly MasterMix (NEB). The PAX3-FOXO1 cDNA was synthesized using a BioXP system (Synthetic Biosystems, Inc., at the Center for Advanced Technologies, UCSF) and cloned into pBABE-Neo with a Gibson Assembly MasterMix (NEB).

Human UBA5 and UFM1 were cloned into pET15b and UFC1 was cloned into pET32a as previously described^{20 (link)}. A UFC1 construct called UFC1-NC was generated by mutating C69 and C165 to serine leaving only the active site C116. In UBA5(Δ349-389) the missing residues were replaced by a short sequence of GSG. The fusion constructs UFC1-UBA5(389-404) and UBA5(347-404)-UFC1 were generated using Gibson assembly (Gibson assembly master mix, New England Biolabs) according to the manufacturer’s protocol. The point mutants of UBA5 and UFC1 were generated by Pfu Ultra II Fusion HS DNA polymerase, an upgraded fusion polymerase technology developed by Agilent.

Editing of the LATE gene was performed using the CRISPR/Cas9 system. Guide RNA sequences were amplified with primer sets (Supplementary Table S1) and pGTR (Xie et al. 2015 (link)) as a template, and then the amplified fragments were cloned into the AarI site of the pKI1.1R vector (Tsutsui and Higashiyama 2017 (link)) using Gibson Assembly® Master Mix (New England BioLabs). Using this vector, Arabidopsis plants were transformed via Agrobacterium in accordance with the floral dip method. The T₁ generation of transgenic plants was screened on MS medium containing 30 mg l⁻¹ hygromycin and 250 mg l⁻¹ vancomycin, followed by transfer into the soil. Successful genome editing of the plants was confirmed by PCR and sequence analysis. Those individuals that were homozygous at the editing sites of the LATE gene and Cas9-free were selected from the T₃ progeny. These plants were grown at 22°C under long-day and short-day conditions. Flowering time was quantified by counting the total leaf number when the main inflorescence stem reached to about 5 cm and the number of days after sowing until flower bud formation is confirmed. Statistical analysis comparing WT and genome-edited plants was performed using Student’s t-test (p<0.05).

To construct the vector harbouring the CRISPRa system (pJDE003), the CRISPRi (KRAB-dCas9-MeCP2) gene fusion of pGC02 ^{1 (link)} was replaced with dCas9-VPR cassette, which was PCR amplified (Q5 High-Fidelity 2X Master Mix, NEB M0492L) from the plasmid pCC_05 ^{2 (link)} with primers oJDE005 and oJDE006 following instructions from manufacturer. pGC02 was digested with XbaI-FD and BamHI-FD (Thermo Fisher FD0685 and FD0054) and sequentially dephosphorylated with FastAP (Thermo Fisher EF0651) following the manufacturer’s recommendations. The digested pGC02 vector and the PCR insert with the CRISPRa system (previously treated with a 15 min DpnI enzyme incubation, Thermo Fisher FD1704) were assembled by Gibson assembly using a 2:1 insert:vector ratio with Gibson Assembly Master Mix (NEB E2611S). Assemblies were transformed into NEB 5-alpha E.coli competent cells and single colonies were picked and sequence validated by Sanger sequencing. Frozen stock of the correct construct cells were regrown for plasmid Maxiprep extraction (QIAGEN 12362) for subsequent virus production.

The type stain DSM17834 of the delta-proteobacterium P. borbori was purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). It was cultivated aerobically in PME medium (0.5% peptone, 0.3% meat extract, pH 7.0) at 28 °C, and a growth curve of biological triplicates was recorded. The generation time (G) during exponential growth was estimated using the formula $G=\frac{\mathrm{\Delta }t}{3.3\log \left(\frac{{\mathrm{OD}}_2}{{\mathrm{OD}}_1}\right)}$ .
Protein fusions for in vivo localization with epi-fluorescence microscopy were generated by PCR amplification of the frpl gene of P. borbori including 200 bp of the 5′ untranslated region and insertion into pSG1164 vectors with an N- or C-terminal mVenus coding sequence and a ‘GGGGGSL’ linker sequence in frame using Gibson Assembly Master Mix (NEB). Correct assembly was verified by Sanger Sequencing (Microsynth). Chemically competent P. borbori were prepared by modification of a protocol by Irani and John^{53 (link)}, initially developed for P. aeruginosa, as follows: the medium was changed to PME, and temperatures were lowered to 28 °C. Plasmids were transformed into P. borbori following the transformation protocol of Irani and John^{53 (link)}, but changing the heat shock temperature to 30 °C, the medium to PME, the growth temperature to 28 °C and the carbenicillin concentration to 100 µg ml⁻¹. Plates were incubated at 28 °C for 48 h until colonies were visible.