Extended Data Fig. 10: Applications of NovaIscB, related to Fig. 5.

(A) A-to-G conversion efficiency of OrufIscB-KRK-ABE8e at 10 target sites in HEK293FT cells. Data are presented as mean +/- SD, n = 4 replicate transfections. (B) Distribution of the number of guides targeting each human CDS for knockdown by OrufIscB/NovaIscB using an NTAAA TAM. (C) Normalized target transcript levels in HEK293FT cells co-transfected with CRISPRoff or OMEGAoff plasmids and negative control (AAVS1 targeting) guides or guides targeting NP63 and PCSK9 as assayed by RT-qPCR. Data are presented as mean +/- SD, n = 4 replicate transfections. (D) Normalized target (FABP4) transcript levels in HEK293FT cells transfected with plasmids or in vitro transcribed RNAs encoding ωRNAs and OMEGAoff as assayed by RT-qPCR. Data are presented as mean +/- SD, n = 4 replicate transfections. (E) DNA methylation levels at CpG sites in the targeted FABP4 promoter region assayed by bisulfite sequencing (BS-Seq) in HEK293FT cells co-transfected with OMEGAoff plasmids and negative control (AAVS1 targeting) ωRNA or FABP4 targeting ωRNA. Data are presented as mean +/- SD, n = 3 replicate transfections. The amplicon sequence for BS-Seq is included. The guide sequences are labeled as red. The two CpG sites are labeled as blue. Gray shading denotes primer binding regions to make the amplicon. (F) Normalized target transcript levels in HEK293FT cells at time points from 1 to 3 weeks after co-transfection of CRISPRoff or OMEGAoff plasmids and guides targeting FABP4 or ASCL1 relative to an AAVS1 targeting negative control guide as assayed by RT-qPCR. Data are presented as mean +/- SD, n = 4 replicate transfections. (G) Normalized target (ASCL1) transcript levels in HEK293FT cells transfected with various plasmid amounts of OMEGAoff or CRISPRoff and associated guide RNAs as assayed by RT-qPCR. Data are presented as mean +/- SD, n = 4 replicate transfections. (H) Normalized target transcript levels in HEK293FT cells co-transfected with dCas9-VPR or dOrufIscB-KRK-VPR plasmids and negative control (AAVS1 targeting) guides or guides targeting ASCL1, CA2 or CALD1 as assayed by RT-qPCR. Data are presented as mean +/- SD, n = 4 replicate transfections. (I) Quantification of PCSK9 protein levels assayed by western blot in AML12 cells after AAV infection of the OMEGAoff targeting Rosa26 (non-targeting) or Pcsk9. Assays were conducted 1 week post infection with different volumes of viruses as indicated, related to Fig. 5G. (J) RNA-seq analysis of HEK293FT cells co-transfected with OMEGAoff or CRISPRoff plasmids and guides targeting CLTA or CALD1 relative to untransfected. The comparison of target guides vs negative control was conducted by DESeq2 and displayed as volcano plots. The differential expressed transcripts (-Log10P-value > 5.5, Log2 fold change < -1 or > 1) were labeled, n = 2 replicate transfections. (K) Liver function tests (total Bilirubin and ALT assays) of serum samples from uninjected, PBS injected, Rosa26-targeting, and Pcsk9-targeting ωRNAs injected mice at the time point of 24 weeks (6 months) after injection. Data are presented as mean +/- SD, n = 4 animals.