Fig. 5: NovaIscB is a platform for compact genome and epigenome modification.
From: Evolution-guided protein design of IscB for persistent epigenome editing in vivo
a, Schematic of AAV packaging capacity when using NovaIscB. EFS, a promoter to drive the expression of protein components. b, Distribution of the number of NovaIscB-compatible NTAAA TAM sites within 500 bp of a transcription start site of all human coding genes. TSS, transcription start site. c, Left: various protein fusion architectures tested in the OMEGAoff system. Right: Normalized transcript levels of the MYL6 transcript in HEK293FT cells measured by RT-qPCR for each corresponding fusion architecture with NT (AAVS1 targeting) or T (MYL6 targeting) guides. Data are presented as mean ± s.d.; n = 3 replicate transfections. d, Normalized target transcript levels in HEK293FT or AML12 cells co-transfected with CRISPRoff (SpCas9) and OMEGAoff (OrufIscB-KRK) plasmids with negative control (AAVS1 targeting) guides or targeting guides as assayed by RT-qPCR. Data are presented as mean ± s.d.; n = 4 replicate transfections. e, Normalized ASCL1 transcript levels in HEK293FT cells co-transfected with plasmids or in vitro-transcribed RNAs encoding ωRNAs and OMEGAoff (OrufIscB-KRK) as assayed by RT-qPCR. Data are presented as mean ± s.d.; n = 4 replicate transfections. f,g, Normalized Pcsk9 transcript levels measured by RT-qPCR (f) and PCSK9 protein levels assayed by western blot (g) in AML12 cells 1 week after AAV transduction of OMEGAoff (NovaIscB) targeting Rosa26 (nontargeting) or Pcsk9 (targeting). Data are presented as mean ± s.d. The error bars denote s.d.; n = 3 replicate transductions. h, Experimental workflow for in vivo Pcsk9 repression using AAV delivery of OMEGAoff (NovaIscB). i, Time course of serum PCSK9 (top) and total cholesterol (bottom) levels in mice. The error bars denote s.d.; n = 4 animals for each condition across all time points.
