Sgrnas

Sequences used to build the gene model cassettes and repair templates were amplified from the Manes.05G046900 locus of cassava cultivar TME 7 genomic DNA with Q5 DNA polymerase (New England Biolabs). The Cas9 protein was previously described (Fauser et al., 2014) and was expressed from a double 35s promoter. sgRNAs (Jinek et al., 2012) #7 and #11 were synthesized (Integrated DNA Technologies, CA, USA) and cloned by Golden Gate Assembly (Engler et al., 2009) under control of the Arabidopsis thaliana U6 (Li et al., 2013) and 7SL (Baltes et al., 2015) promoters, respectively. Vectors were constructed primarily by Gibson Assembly (Gibson et al., 2009) from PCR amplicon and plasmid components. The BeYDV GVR was described previously (Baltes et al., 2014; Čermák et al., 2015). Agrobacterium tumefacien strain LBA4404 harbouring pCAMBIA2300‐based binary vectors (GenBank: AF234315) was used for the transformation of cassava.

Plasmid pIRL58 (Addgene, 166886) bearing the Streptococcus thermophilus CRISPR–dCas9 system (dCas9_Sth1)^{13 (link)} was used to modulate the RNA expression level of target genes in Mtb mc²6206 cells. Oligonucleotides for single guide RNAs (sgRNAs; Integrated DNA Technologies) were cloned into pIRL58. After verification by Sanger sequencing, pIRL58 and pIRL19 (Addgene, 163634, which supplied the L5 integrase function on a separate suicide vector) were co-transformed into Mtb cells by electroporation using GenePulser (BioRad) at 2,500 V, 700 Ω, and 25 μF. Single colonies were picked from the solid culture plates with 20 μg ml⁻¹ kanamycin (Goldbio, K-120) selection after 14–21 days of culture. Target gene knockdown was induced by adding 100 ng ml⁻¹ ATc (Sigma, 37919). The sgRNA and primer sequences are listed in Supplementary Table 5.