Ultra gelred

The pegRNAs or xr-pegRNAs were in vitro transcribed from T7 promoter-led templates using the MEGAshortscript™ T7 kit (Invitrogen). Their degradation profile under an endonuclease-inhibited condition was analyzed as recently described¹⁹ , with minor modifications. Briefly, the nuclear extracts were prepared from near-confluent HEK293T cells using the ExKine™ kit (Abbkine). One μg of in vitro-transcribed RNA was added to each sample that contained ±1.5 μl of fresh nuclear lysate in a total volume of 10 μl of reaction buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 50 mM NaCl, 2 mM DTT, 1 mM NTP and 0.8 U μl⁻¹ of RNaseOUT [from Vazyme] endonucleases inhibitor). After incubation at 37 °C for 20 min, the samples were resolved on 2.0% agarose gels and stained with Ultra GelRed (Vazyme). The intensities of the bands were quantitated by ImageJ (1.53i). The uncropped pictures of the gels are provided in the Source Data file. As a positive control of degradation protection, 1 μg of RNA samples were pre-incubated with (or without) 5 μg Cas9 protein at the room temperature for 10 min. The samples were next subjected to incubation with 3 μl of nuclear lysate in the same condition as above. In all, 2 μl 10 U μl⁻¹ of protease K solution was then added to terminate the reaction. The remaining RNA was precipitated using isopropyl alcohol and subjected to RT–qPCR analyses.

Genomic DNA was isolated from the young seedlings of the tested genotypes using the protocol in Sharp et al. (1988) (link) with minor modification. Specific primers of 23 reported Pm genes (Pm1-5, Pm6, Pm8, Pm12, Pm21, Pm24, Pm35, Pm37, Pm41, Pm42, Pm45, Pm47, Pm52, Pm58, Pm59, Pm60, Pm61, Pm68, and Pm69) were used to test all 137 wheat accessions in this study (Supplementary Table S2). PCR amplification was performed according to Jin et al. (2023) (link). PCR products of most primers were detected in 8% non-denaturing polyacrylamide gels with 19:1, 29:1, or 39:1 ratios of acrylamide and bis-acrylamide, and were silver stained (Santos et al., 1993 (link)). The rest of the PCR products were separated by agarose gel with a concentration of 1.5% with nucleic acid dyestuff ultra GelRed (Vazyme Biotech Co., Ltd. China), and were then observed using the Gel Documentation System (Gel Doc XR+, BIO-RAD, Hercules, CA, United States) (Gebrewahid et al., 2020 (link)). See Supplementary Table S2 for the marker information and corresponding positive control of tested Pm genes.

DNA was extracted from LLC-OVA cell lines utilizing the FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme, DC102-01). PCR amplifications were then conducted using specific primers, ensuring equivalent DNA quantities in each PCR reaction.
A 10 μl PCR mixture was prepared, comprising 1 μl of genomic DNA, 0.5 μl of each primer, 5 μl of 2 × Taq Master Mix (Vazyme, P112-01), and 3.5 μl of DNase/RNase-free ddH2O. The PCR parameters were set as follows: initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 35 s, and extension at 72 °C for 35 s. The reaction was concluded with a final extension at 72 °C for 5 min.
Following amplification, the PCR products were subjected to electrophoresis on a 1.0% agarose gel stained with Ultra GelRed (Vazyme, GR501-01) and visualized under ultraviolet light. The DL5000 DNA marker (Vazyme, MD102-01) was used to determine the molecular weight of the PCR products. The primers of OVA257-264^{102 (link)} were synthesized by Tsingke Biotechnology Co., Ltd (Beijing, China). The sequences of the primers are listed in Supplementary Table 2.