Mix2seq

Plasmid pDHL1029-mScarlet (pMV371) was constructed as follows; primers are listed in Table S1. First the mScarlet gene was PCR amplified from pmScarlet-C1 using oMV1367 and oMV1368, after which the resulting PCR product was digested with EcoRI and XmaI. Next the pDHL1029-msfGFP backbone was PCR amplified using oMV1373 and oMV1374, after which the resulting PCR product was digested with EcoRI and XmaI. Finally, the fragments were mixed together and ligated to make pDHL1029-mScarlet (pMV371). The final plasmid was PCR and sequence verified. All enzymes used were supplied from NEB.
All of the remaining plasmids created in this study (pCC1FOS derivatives) were constructed using λ red mediated homologous recombination [75 (link)], using exactly the same protocol as described above, in strain EPI300 (Lucigen). Correct integration of PCR products was further verified by sequencing (Eurofins, Mix2Seq). All primer sequences for constructing the pCC1FOS derivatives are listed in Table S1. The frt-flanked antibiotic cassette could be removed as previously described [76 (link)] and using the protocol described above. All the different loci targeted were PCR-validated and sequence-verified. Correct constructs were isolated from EPI300 after copy control induction, and transformed to the relevant background for competition assays.

PCR was performed using Q5® High-Fidelity DNA Polymerase (M0491, NEB) with DNA oligos from Integrated DNA Technologies (IDT). PCR products were gel purified using Gel Purification Kit (D4002, Zymo Research). To generate entry vectors, DNA fragments were inserted into BsaI-digested GreenGate empty entry vectors [52 (link)] via Gibson assembly (2× NEBuilder Hifi DNA Assembly Mix, NEB) or restriction ligation with T4 DNA ligase (NEB). Base editors, gRNAs, and fluorescent reporter vectors were assembled using Golden Gate cloning (30 cycles (37°C, 5 min; 16°C, 5 min); 50°C for 5 min; 80°C for 5 min) with BsaI or BbsI [52 (link)]. Vectors were transformed by heat-shock transformation into DH5α E.coli or One Shot™ ccdB Survival™ competent cells (Thermo Fisher Scientific). Cells were plated on lysogeny broth medium containing 100 μg mL^-1 carbenicillin, 100 μg mL⁻¹ spectinomycin, 25 μg mL⁻¹ kanamycin, or 40 μg mL⁻¹ gentamycin, depending on the selectable marker. Plasmids were isolated (GeneJET Plasmid Miniprep kit, Thermo Fisher Scientific) and confirmed by restriction enzyme digestion and/or Sanger sequencing (Eurofins, Mix2seq). All plasmids are described in Additional file 3. All primers are listed in Additional file 4.

Plant material was harvested for DNA extraction with the CTAB method [73 ]. Either the first true leaf pairs or entire seedlings were harvested, depending if the material was upscaled or not. A region around the CRISPR/Cas target site was PCR amplified using ALLin Red Taq Master Mix, 2X (highQu). The PCR products were analysed via agarose gel electrophoresis and purified by bead purification with HighPrep PCR (MAGBIO). The purified samples were sent for Sanger sequencing (Mix2seq; Eurofins Scientific) and analysed using ICE (https://ice.synthego.com/#/) and/or EDITR [41 (link)]. The ICE KO-score represents the proportion of cells that have either a frameshift or 21+ bp indel. See Supporting Tables for the list of primer and target sequences. The number of individuals analysed is specified for each experiment.