Fig. 4

Assessment of viral DNA accumulation, relative MeE3L expression, and predicted MeE3L primary structure in transformed cassava protoplasts. a Relative DNA accumulation of SACMV- and SACMV + CRISPR-Cas9-transformed cassava protoplasts under different transformation conditions. ΔMeE3L = mutant CRISPR-edited MeE3L. Real-time qPCR was performed in triplicate using DpnI-treated total DNA extracted from cassava protoplasts 24 hpt as template. b MeE3L relative expression levels in transformed cassava protoplasts. V = SACMV-transformed. ΔMeE3L = gene-edited MeE3L. C* = transformed with CRISPR construct lackingt gRNA duplex. RT-qPCR was performed in triplicate using total mRNA as template. c Stop mutation induced in SACMV-infected susceptible T200 MeE3L. d Stop mutation induced in SACMV-infected tolerant TME3 MeE3L. e The predicted amino acid sequence of tolerant TME3 MeE3L at reference sequence positions 2–148 showing multiple mutations in SACMV-infected variant. V = SACMV-infected. C = gene-edited. Sequence alignment was conducted in MEGA-X [40]. f Frequency and types of mutation at target gRNA sites from CRISPR-transformed protoplasts. Frequency of clones with altered sequence was obtained by expressing number of amplicons from a polyclonal mix with sequence alteration as a ratio of total amplicons (n = 10 per genotype) sequenced. Mutations were determined by aligning amplicon sequences with wild-type reference AM560-2 [14] and TME3 (RefSeq ID: RSFT01000007,GenBankassembly GCA_003957995.1 (unpublished data)) MeE3L homologs. Alignment was conducted on MEGA-X [40] using the CLUSTAL W algorithm for multiple sequence alignment [44]. g Timeline for rapid screening of genes associated with the response to South African cassava mosaic virus in cassava