All Sanger DNA sequencing confirmation in this study was performed with the indicated sequencing primers at the Research Technology Support Facility Genomics Core, Michigan State University, East Lansing, MI.

For proSpASFF1::SpASFF1 ORF (pK7WG), a 1.8-kb region of the upstream region and ORF of SpASFF1 was split into four amplicons using four sets of primers: ASFF_001_F/R, ASFF 002_F/R, ASFF_003_F/R, and ASFF_004_F/R (table S3). The first and fourth amplicon contained adapters for assembly into pENTR-D-TOPO that has been digested with NotI/AscI, respectively. The construct was assembled using New England Biolabs (NEB) Gibson Assembly according to manufacturer specifications (NEB, Ipswich, MA). The construct was verified by Sanger sequencing using M13 Forward, T7 promoter primers, and cloning primers. The insert was subcloned into pK7WG (54) using the LR Clonase II Enzyme mix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Presence of the insert was determined by colony PCR using ASFF_001F/R. Completed vectors were transformed into Agrobacterium strain AGL0. Leaf material from recovered plants were archived on FTA PlantSaver cards (GE Healthcare, Uppsala, Sweden) and genotyped by PCR amplification with GoTaq Green Mastermix and pK7WG-Kan-F/R primers (table S3).

For proSpASFF1::GFP/GUS (pKGWFS7), a 1.8-kb region of the upstream region of ASFF1 was amplified from S. pennellii LA0716 genomic DNA using ASFF_promoter_F1/R1 primers (table S3). pENTR-D-TOPO was digested with Not I/Asc I to linearize the vector and create overhangs compatible for Gibson Assembly. The amplicon also contained adapters for insertion into pENTR-D-TOPO digested with Not I/Asc I. Constructs were Sanger-sequenced using M13F/R primers in addition to the ASFF_promoter_F1/R1 primers. LR Clonase II Enzyme mix was used to subclone the fragment into pKGWFS7 (54). The construct was transformed into AGLO for plant transformation using the described protocol.

The CRISPR-ASFF1 vector was constructed as follows. CRISPR sgRNAs were designed using the site finder toolset in Geneious v10 (www.geneious.com). Two target sequences located on the exon were selected for their high on-target activity scores, based on a published algorithm (55), and low off-target scores against published S. pennellii genome database (56). Each sgRNA was obtained as a gBlock synthesized in vitro by IDT (Integrated DNA technologies) (www.idtdna.com) (table S3) and subsequently assembled with pICH47742::2x35S-5′UTR-hCas9(STOP)-NOST [Addgene plasmid no. 49771, provided by S. Kamoun (Sainsbury Lab, Norwich, UK)] (57), pICH41780 (Addgene plasmid no. 48019) and pAGM4723 (Addgene plasmid no. 48015, both gifts from S. Marillonnet) (58), and pICSL11024 [Addgene plasmid no. 51144, a gift from J. D. Jones (Sainsbury Lab, Norwich, UK)] using Golden Gate Assembly. In short, the restriction-ligation reactions (20 μl) were set up by mixing 15 ng of synthesized sgRNAs with 1.5 μl of T4 ligase buffer (NEB), 320 U of T4 DNA ligase (NEB), 1.5 μl of bovine serum albumin (BSA; 0.1 mg/ml; NEB), 8 U of BpiI (Thermo Fisher Scientific), and 100 to 200 ng of the intact plasmids. The reactions were incubated at 37°C for 30 s, followed by 26 cycles (37°C, 3 min; 16°C, 4 min), and then incubated at 50°C for 5 min and 65°C for 5 min. The ligated products were directly used to transform E. coli competent cells. Positive clones were chosen based on colony PCR and sequenced at the Michigan State University Research Technology Support Facility (MSU RTSF) using the pAGM4723_SeqF1, pAGM4723_SeqR1, pICSL11024_SeqF1, pICH47742CAS9_SeqF2, pICH47742_SeqF1, pICH41780_SeqR1, and ASFF_SeqR primers (table S3). The construct was transformed into S. pennellii LA0716 using the plant transformation protocol described below. Leaf material from recovered plants were archived on FTA PlantSaver cards (GE Healthcare, Uppsala, Sweden) and genotyped by PCR amplification with ASFF_F/R, followed by Sanger sequencing with ASFF_SeqR (table S3).

For spasff1 line transcript analysis, RNA was extracted from spasff1-1-1/1-1-2 lines using RNeasy Plant Mini Kit according to the kit specifications (Qiagen, Venlo, The Netherlands). RNA was quantified using a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA). One microgram of RNA was used for cDNA synthesis using Superscript II Reverse Transcriptase according to the manufacturer’s specifications. The ASFF1_transcript_amp_01F/R primers (final concentration, 0.5 μM) were used to amplify the region within the ASFF1 CDS (coding sequence), which was cloned into pMINI-T 2.0 (NEB, Ipswich, MA). T7 and SP6 promoter primers were used for Sanger sequence confirmation of the inserts, and ClustalW was used for alignment of the transcripts (www.ebi.ac.uk/Tools/msa/clustalo/).

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