For construction of guide RNA genes

For construction of guide RNA genes, two different promoters of the rice small nuclear RNA U6 genes were commercially synthesized based on the genome sequence of the
rice cultivar Nipponbare. For sgRNA constructs, the BtgZI or BsaI cutting site was placed downstream of the U6 promoter and used to fuse the seed sequences to the tracrRNA tail sequences of either 48 nucleotides [sgRNA(+48)] or 85 nucleotides [sgRNA(+85)], the guide RNA architectures based on the work as published (19). Those cassettes were synthesized by GenScript and cloned into pENTR4 (Life Technologies) by BamHI and EcoRI restriction and DNA ligation, individually, resulting in a set of pENTR-sgRNA vectors in which the guide RNA cassettes could be mobilized to pUbi-Cas9 using the Gateway LR clonase (Life Technologies). For the dual tracrRNA and crRNA [dual guide RNA (dgRNA)] constructs, the DNA sequences for tracrRNA and the crRNAdirect repeats were adapted from Jinek’s work (18). The tracrRNA-coding DNA and the rice U6 promoter were synthesized from GenScript; the DNA for the crRNA repeat sequence and another rice U6 promoter was also synthesized from GenScript. The BsaI and BtgZI restriction sites were designed and located within the direct repeats of crRNA for cloning of seed sequences. Both crRNA and tracrRNA cassettes were cloned into pENTR4 in tandem by BamHI and EcoRI. The sequences of those synthetic guide RNAs (sgRNA or dgRNA) are provided in Supplementary data (Supplementary Figure S1).