GSM6595139: Cas12a Base editor 20220912_T1_wheat; Triticum aestivum; OTHER
For genotyping individual wheat transformed plants, DNA was isolated according to Edwards method (Edwards et al., 1991). A piece of leaf (0.5-1 cm), harvested in 1 ml tubes on 96-well plate (VWR, 732-3716) and flash frozen in liquid nitrogen. Two metal beads (3 mm) were added and tissue was ground to powder by shaking the plate at 20 Hz for 1 minute (Retsch, Mixer Mill MM 400). 400 µl of extraction buffer (100 mM Tris-HCl pH 8.0, 500 mM NaCl, 50 mM EDTA, 0.7% SDS) was added to individual samples and incubated 30 min at 60°C. Samples were centrifuged and 300 µl of the supernatant was mixed to 300 µl of isopropanol for DNA precipitation. Samples were then centrifuged and supernatant removed. The pellet was washed with 70% Ethanol,dried at room temperature, anddissolved in 100 µl of 10 mM Tris-HCl pH=8.0.  For sorted material, 2 μl of the solution containing sorted cells was used as template in a 20 µl total reaction volume for amplicon PCR using the Phire Plant Direct PCR Kit (Thermo Fisher Scientific, F160L) according to manufacturer's recommendations.  For nCas9-BE, base editing efficiencies were measured by Sanger sequencing. 500-700 bp amplicons were designed to amplify target sites (Supplementary tables 2-3). 5 µl of the Phire PCR reaction was verified on a 1% agarose gel (Ultra-Pure agarose, Invitrogen, # 16500-500), with a 1kb bench top ladder (Promega, G754A). 15 µl was purified using 1.8× HighPrep™ PCR beads (MAGBIO Genomics, AC-60500), eluted in 35 μl 10mM Tris-HCl pH 8.0 and sent for Sanger sequencing (Eurofins, Mix2Seq). Chromatograms were processed using EditR to obtain base editing efficiencies (Kluesner et al., 2018). Statistical tests were conducted with GraphPad Prism 9.0.0 or VassarStats online tool (http://vassarstats.net/).    For dCas12-BE and nuclease-active LbCas12a, base editing and indel efficiencies were measured using NGS. 210-260 bp amplicons were designed to amplify target sites. 6-nt indices (Faircloth and Glenn, 2012) were added to forward and reverse primers for pooling and demultiplexing amplicon reads after sequencing (Supplemental table 2). 5 µl of the Phire PCR reaction was verified on a 2% agarose gel with a low molecular weight ladder (NEB, no. N3233S). 15 µl of PCR products were pooled and purified using PCR Purification Kit (Zymo Research Co., D4013). Depending on the amplicon, an extra gel purification was conducted (Zymo Research, D4002) to specifically isolate PCR band of the target site. The DNA concentration was measured with Qubit (Invitrogen) according to manufacturer's protocol and adjusted to 2 ng µl-1. Paired end sequencing was performed with Eurofins NGSelect amplicons (5M reads 2x150bp). Reads were demultiplexed using Je-demultiplex (Girardot et al., 2016) and individual .fastq files were obtained as previously described (Bollier et al., 2020) using a Galaxy workflow (https://usegalaxy.be). Demultiplexing statistics can be found in Supplemental data 2. Library was generated at Eurofins Genomics using NGSelect Amplicon Adaptor Ligation for 5M reads 2x150bp. Amplicon-seq