The multigene binary cassettes were assembled using the Golden Gate cloning system (54, 55). Level 0 (L0) modules were assembled for four transcription units [termed level 1 (L1) modules]. Following digestion of L0 modules with Bsa I and ligation with T4 ligase, the hptII gene (hygromycin B resistance gene) was inserted between the CaMV35S promoter and terminator sequences to create the L1-F1-p35S-hptII-t35 vector required for plant selection. Similarly, a L1 module termed L1-F2-p35S-Cas9-eGFP-NLS-tHSP transcription unit was assembled from synthesized L0 modules. These L0 modules comprised the pCaMV35S promoter, a Cas9 derived from Streptococcus pyogenes codon optimized for expression in cassava, the eGFP reporter gene fused in-frame to a nucleoplasmin NLS, and a terminator sequence of heat shock protein (tHSP) (56). This L1 module was used for protoplast transfection. The sgRNA transcript unit (psynU6-sgRNA-Scaffold-Terminator) was synthesized in its entirety and cloned into a L1-F3 backbone vector to generate L1-F3-psynU6-sgRNA-Scaffold-Terminator. psynU6 is a synthetic promoter using the consensus sequence of the three U6 variants in Arabidopsis (10) and fused to a chimeric scaffold (9). The incorporated GBSSsgRNA4 sequence was 5′-GCTTGGGATACCTCTGTAT-3′, whereas for PTSTsgRNA2, it was 5′-GAATATCCTACGGTTGGCGA-3′. The fourth and final transcriptional unit used L0 modules of the pCaMV35S promoter, the FT coding sequence from Arabidopsis (17), and the Agrobacterium nopaline synthase (nos) terminator to create L1-F4-pCaMV35S-AtFT-tNOS. One-step Bpi I restriction enzyme digestion and ligation of the four L1 transcriptional units, a L2 backbone vector, and an endlinker (ELE-4) resulted in assembly of pCas9-sgGBSS-FT and pCas9-sgPTST-FT. Both expression plasmids were sequenced to verify the integrity of the cloned units. Multiple PAMs comprising the NGG nucleotide triplet were identified in the GBSS sequence of cv. 60444, and each was assessed by Basic Local Alignment Search Tool (BLAST) analysis and in silico profiling to determine suitability for gene disruption with minimal off-target effects. GBSSsgRNA4 targeting sequence in exon 2 and approximately 470 bp downstream of the transcription start site was selected for in vitro analysis. For PTST1, PTSTsgRNA2 was selected to target an exon at the 3′-end of the coding sequence. This region encodes a CBM48 domain required for interaction of PTST1 with starch (36); site-directed mutagenesis of this domain in Arabidopsis PTST1 abolishes starch binding (35).

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