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. 2023 Jul 13;14:1225775. doi: 10.3389/fpls.2023.1225775

Figure 1.

Figure 1

In vitro validation of sgRNA cleavage activity and approach for targeted mutagenesis of bahiagrass MgCh. (A) In vitro sgRNA cleavage assay; a 659 bp amplicon is cut into 472 and 187 bp fragments by the Cas9-191r ribonucleoprotein (RNP), 413 and 246 bp fragments by the Cas9-250r (sgRNA1) RNP, 384 and 275 bp fragments by the Cas9-279f RNP, and 346 and 313 bp fragments by the Cas9-350f (sgRNA2) RNP. (B) 586 bp amplicon used for mutation screening; protospacer adjacent motifs (PAMs) are indicated with red letters, indels at sgRNA targets eliminate PvuII and KflI sites. (C) 11.125 kb fragment delivered for targeted mutagenesis of MgCh, including O. sativa U6-sgRNA expression cassettes, nptII selectable marker with Z. mays ALS2 promoter and S. bicolor HSP16.9 terminator, and Cas9 with N- and C-terminal nuclear localization signals (NLSs) driven by ZmUbi promoter with A. thaliana HSP18.2 terminator.