Plant materials and growth conditions
Environmental conditions during seed production, as well as during seed storage, can affect seed vigour. Therefore, all experiments were conducted with wild-type and transgenic Arabidopsis thaliana (Columbia ecotype, Col-0) seeds and, within the same experiment, harvested from plants grown side by side. All transgenic lines were generated by the authors and not obtained commercially. For growth experiments, plants were grown in vitro on ½ Murashige and Skoog (MS) medium  supplemented with 1% sucrose at 21°C under a 16-h day/8-h night regime (75µM; Spectralux Plus NL-T8 36W/840/G13 fluorescent lamp). For stress-inducing conditions, the medium was supplemented with Mannitol (Sigma, 25mM or 50mM), NaCl (ChemLab, 50mM or 100mM), Bleomycin Sulfate (Sigma, 0,3µg/mL) or Hydroxyurea (Sigma, 0,75mM). For the application of heat stress, plates were transferred to a common 37°C incubator for 24h. For UV-stress, we treated the transgenic lines with a continuous low-dose stress (40 W/m2 light supplemented with 0,42 W/m2 UV) for one, two or three days. For lines targeting ADH1, seeds were pre-treated with 30mM Allyl alcohol (Sigma) for two hours before sowing. All stress conditions were imposed immediately after stratification unless noted otherwise. The HDR reporter vector line pK LacI-AtCas9 was previously described .
Plasmid constructs and plant transformation
All cloning reactions were transformed via heat-shock transformation into ccdB-sensitive DH5α Escherichia coli or One Shot ccdB Survival 2 T1R Competent Cells (Thermo Fisher Scientific). Colonies were verified via colony-touch PCR, restriction digestion, and/or Sanger sequencing by Eurofins Scientific using the Mix2Seq service. All PCR reactions for cloning were performed with Q5 High-Fidelity DNA Polymerase (New England Biolabs). Gibson assembly reactions were performed using 2× NEBuilder Hifi DNA Assembly Mix (New England Biolabs). Column and gel purifications were performed with Zymo-Spin II columns (Zymo Research). Golden Gate entry modules were constructed by PCR amplification of gene fragments and inserting the purified PCR product into a BsaI-digested GreenGate entry vector [32, 37] via restriction-ligation using BsaI (New England Biolabs) or Gibson assembly. All generated clones were verified via Sanger sequencing. See Tables S1-3 for the list of primer and target sequences, a complete list of plasmids and cloning primers.
AsCas12a, FnCas12a, and LbCas12a were amplified from pY010, pY004, and pY016, respectively and cloned into pGGC000 using restriction ligation. pY010, pY004, and pY016 were gifts from Feng Zhang (Addgene plasmids # 69982, # 69976, and # 69988; ).
Cas12a expression vectors were assembled with Golden Gate cloning by combining pGG-A-PcUBI-B, pGG-B-Linker-C, pGG-C-Cas12a-D, pGG-D-linker-E, pGG-E-G7T-F, pGG-F-LinkerII-G into pFASTRK-AG and verified with restriction digest with PvuI and NotI. The Golden Gate destination module (A-ccdB/CmR-G) was inserted into correct plasmids via HindIII digestion and Gibson assembly as previously described  and confirmed with restriction digest with PvuI and NotI.
crRNA entry vectors were created using a gBlock (IDT) template containing HH and HDV ribozyme sequences with a constant Cas12a scaffold sequence and a pair of BbsI restriction sites to add novel crRNA sequences. The gBlock fragment was PCR amplified with primers flanked by BsaI restriction sites and cloned into pGGB000 via restriction ligation to create pGG-B-HH-Cas12ascaffold-HDV-C. crRNAs were designed with Geneious R11  with a length of 24nt and cloned into pGG-B-HH-Cas12a-HDV-C with homolog-specific scaffolds (AsCas12a: CTTGTAGAT; FnCas12a: GTTGTAGAT; LbCas12a: AAGTGTAGAT; ) using annealed oligo cloning with BbsI . Entry vectors were cloned into Cas12a destination vectors with pGG-A-pRPS5A-B , pGG-B-crRNA-C, pGG-C-linker-D, pGG-D-pea3AT-G and validated with restriction digest with NdeI.
PDS3 Cas9 gRNAs for the promoter and tobacco tests were cloned into pEn_Chimera  via annealed oligo cloning. The ZmUbi, RPS5a, and 35S Cas9 entry vectors were assembled with GoldenGate cloning as previously described for pEN-L4-PcUBI-Cas9-G7T-R1 . The Cas9 entry vectors, pEn_Chimera gRNAs and pBm42GW,3  were recombined with Multisite Gateway as previously described  and the resulting plasmids were confirmed via restriction digestion with NdeI.
pFASTRK-AtCas9-AtU6-Scaffold and pFASTGK-AtCas9-AtU6-Scaffold were created by Gateway Multisite assembly with pFASTRK24GW or pFASTGK24GW , pEN-L4-PcUBI-Cas9-G7T-R1, and pEN-L1-AtU6-26-BsaI-L2  and confirmed with restriction digest with NdeI and HpaI. The PDS3, ADH1, GL1-1, GL1-2, AT2G22460, IM1, VAR1-1, VAR1-2, TIR1-1 and AT4G12990 gRNAs were added to pFASTRK-AtCas9-AtU6-Scaffold via annealed oligo cloning with BsaI and confirmed by restriction digest with NheI and Sanger sequencing of the gRNA. The large deletion vectors were made by adding paired gRNAs via a PCR approach  to pFASTGK-AtCas9-AtU6-Scaffold.
pFASTRK-CBE was created by performing a GoldenGate reaction with pGG-A-PcUBI-B, pGG-B-APOBEC-(GGS)5-C , pGG-C-Cas9-D10A-D, pGG-D-UGI-NLS-E, pGG-E-G7T-F, pGG-F-AtU6-26-AarI-AarI-G into pFASTRK-AG and the AarI site replaced with BsaI-ccdB/CmR-BsaI as previously described . The PDS3-7 and PDS3-9 gRNAs were added to pFASTRK-CBE via annealed oligo cloning with BsaI and confirmed by restriction digest with NheI and BamHI and Sanger sequencing of the gRNA.
The PPD2 gRNAs were added to a Cas9-VQR variant as previously described .
Plant vectors were transformed in Agrobacterium tumefaciens C58C1 by electroporation and transformation of Arabidopsis thaliana was performed via floral-dip . For the construct containing the FASTR or FASTG screenable marker , T1 transgenic seeds were selected under a fluorescence stereomicroscope (Leica M165FC). Non-fluorescent T2 lines were assumed to be T-DNA free. Unless specified otherwise, segregating T2 lines used in growth experiments were not checked for homozygosity or single-locus insertion of the transgene. Transformation of Nicotiana tabacum SR-1 was done by cocultivation of leaf explants with Agrobacterium .
DNA extraction, PCR and sequencing analyses
Plant material was harvested for DNA extraction with the CTAB method . 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 . See Supporting Tables for the list of primer and target sequences. The number of individuals analysed is specified for each experiment.