Fig. 2

In vitro assay to evaluate cleavage efficiency by the Guide-it sgRNA screening kit. (a) PCR products from the amplification of EgFAD2 (lane 1: sgRNA1, lane 2: sgRNA2) and EgPAT (lane 3: sgRNA3, lane 4: sgRNA4) sgRNA templates at 130 bp for in vitro transcription. (b) PCR products are amplified from oil palm genomic DNA containing sgRNA target sites; the PCR templates of candidate sgRNAs are then in vitro combined with a recombinant Cas9 for a fragment cleavage site (c). EgFAD2-T1 (lane 1), EgFAD2-T2 (lane 2), EgPAT-T1 (lane 3), and EgPAT-T2 (lane 4) represent the PCR fragments with 921 bp, 986 bp, 903 bp, and 978 bp length, respectively. Cleavage fragments were present for all sgRNAs; they were cleaved into two fragments by the sgRNA/Cas9 complex: 503 bp and 395 bp for EgFAD2-T1; 559 bp and 404 bp for EgFAD2-T2; 531 bp and 349 bp for EgPAT-T1; and 287 bp and 668 bp for EgPAT-T2. Lane C is a control fragment that has a size of 614 bp and cleaved DNA of 350 bp and 264 bp. Lane W is a negative control, and lane M is a 1 kb plus DNA marker (Invitrogen)