Fig. 2

Repression of gfp in Y. lipolytica by dCpf1 and dCas9. a The dCpf1 and dCas9 expression system. The dCpf1 contains mutations of the RuvC1 nuclease domains while the dCas9 contains mutations of both RuvC1 and HNH nuclease domains. b Schematic of RNA polymerase III promoter used in this study and placement of gRNA protospacers on the target gfp gene. SCR1′-tRNA^Gly is the synthetic RNA polymerase III promoter. gRNA is single guide RNAs. polyT is a string of eight thymines, which serves as an RNA polymerase III terminator. Six gRNAs bind to either the non-template DNA strand or the template DNA strand of ORF and four gRNAs bind to different regions around the promoter. c Microscopic images of the interfered strains with gfp gene by dCpf1. d Microscopic images of the interfered strains with gfp gene by dCas9. e CRISPRi repression of gfp with dCpf1 complexed with ten gRNAs targeting different regions. The control (g0) shows fluorescence of the cells with dCpf1 protein but without the gRNA. f CRISPRi repression of gfp with dCas9 complexed with ten gRNAs targeting different regions. The control (g0) shows fluorescence of the cells with dCas9 protein but without the gRNA. g Characterization of the gfp gene’s expression level of each strain interfered by dCpf1. h Characterization of the gfp gene’s expression level of each strain interfered by dCas9. The error bars (mean ± SD) were derived from triplicate experiments for each strain