Genomic DNA was used as the template in PCR amplification of PTST1 and GBSS spanning the protospacer target site. Oligonucleotide primers specific to PTST1 and GBSS and incorporating PacBio barcodes were synthesized. Reaction conditions were 1× Q5 reaction buffer, 200 μM dNTPs, 0.5 μM PTST-F-BCn or GBSS-F-BCn, 0.5 μM PTST-R-BCn or GBSS-R-BCn, 200 ng of genomic DNA, 1 U of Q5 High-Fidelity DNA Polymerase (New England Biolabs), and sterile nuclease-free water. Conditions were as follows: initial step at 98°C (1 min 30 s); then 25 cycles of 98°C (10 s), 62°C (10 s), and 72°C (20 s); and a final step of 72°C for 2 min. Reactions were cleaned using the Wizard SV Gel and PCR Clean-Up System protocol (Promega) before quantification using the Quant-iT dsDNA Assay Kit (Invitrogen). Reactions were analyzed using PacBio SMRT sequencing [Functional Genomics Centre Zurich (FGCZ)]. To identify indels, the software tool Cas-Analyzer (62) was used in conjunction with PTST1 and GBSS sequences defined via BLASTn (63) on in-house genome data of cassava cv. 60444.

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