Fig. 4.

Properties of modified Cas13 variants. a Schematic of nuclease-dead CasFX (dCasFX) activity. dCasFX carries quadrupl e point mutations that abolish its nuclease activity. As a result, the dCasFX/crRNA complex can be recruited and bind to target transcripts, but it cannot cleave the RNA. b Evaluation of Cas13 cleavage efficiency of dCasFX compared to wild-type CasFX. qPCR data represent expression levels of eCFP. Data were normalized to samples treated with blank crRNA (control). * = p value < 0.05, ** = p value < 0.01, *** = p value < 0.001, p values based on Student’s t test, error bars represent 95% confidence intervals. c eCFP fluorescence when targeted by either CasFX or dCasFX. Nuclei were stained with nuclear green DCS1 (Abcam ab138904). Color was adjusted for color-blind-friendly purpose. eCFP and DsRed fluorescence were measured using their native fluorescence property without using antibody staining. Scale bar = 50 μm. d Schematic of dCasFX for the validation of RNA-protein interactions. dCasFX and crRNA targeting Fer1HCH-RA mRNA were transfected together in one sample. Fer1HCH-RA and IRP1A^C450S, a constitutively RNA-binding form of IRP1A that interacts with the iron-responsive element (IRE) in the Fer1HCH-RA mRNA, were transformed together in a different sample. The two samples were each lysed and combined, followed by immunoprecipitation (IP) of dCasFX (utilizing the attached HA tag) to test for the presence of IRP1A in the pull-down assay. e Western blot showing the IP of dCasFX combined with different crRNAs along Fer1HCH-RA mRNA and the detection of IRP1A in corresponding samples. f Functional schematic of CasFX that carries a mitochondrial localization signal (CasFX^mt). At the N terminus, CasFX^mt is fused with the tim23 mitochondrial signal sequence. Upon binding with crRNA, the complex will localize into mitochondria and target mitochondrial-encoded transcripts. g Mitochondrial localization of CasFX^mt. Nuclei were stained with DAPI (blue) while mitochondria were stained with mitotracker green (Cell signaling 9074S) and CasFX polypeptide was stained with anti-HA antibody (magenta). Scale bar = 25 μm. Color was adjusted for color-blind-friendly purpose. h The relative expression level of mitochondrial-encoded transcripts, COXI and COXII, targeted by RNAi, CasFX^O, and CasFX^mt. Data were normalized to samples treated with no transfected plasmid (control). * = p value < 0.05, ** = p value < 0.01, *** = p value < 0.001, ns = not significant, p values based on Dunnett’s post hoc test, error bars represent 95% confidence intervals. i Western blotting of COXI and COXII when being targeted by RNAi, CasFX^O, and CasFX^mt