Fig. 2: Schematic representation of the screened repressors paired with dSaCas9 and dCjCas9.

a Schematic view of the LV-reporter vector and the parental AAV vector carrying various repressor effectors. LV harbors a dual reporter system consisting of destabilized GFP (dGFP) and Nano-Luciferase (NLuc). Viral long terminal repeats – LTRs; SV40 polyadenylation signal- SV40pA are highlighted.pSV40 drives the expression of the puromycin marker. The vector was transduced at the MOI = 0.2 to enable the selection of the cells carried 1 copy/cell. Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE). The inverted terminal repeats (ITRs) of AAV are highlighted. The EFS-NC promoter that drives the expression of the dCas9-effector (black and red boxes, respectively) is highlighted. Human U6 promoter driving expression of gRNA is highlighted. b Schematic representation of Heterochromatin Protein 1 alpha (HP1a) and Heterochromatin Protein 1 beta (HP1b) The Chromodomain (CD) and Chromoshadow Domain (CSD) are highlighted. The Hinge region separating the CD and CSD domains is highlighted. c Schematic representation of Methyl-Binding Proteins (MBDs). Methyl-Binding Protein 1 (MBD1); Methyl-Binding Protein 2 (MBD2); Methyl-Binding Protein 3 (MBD3); and Methyl-CpG Binding Protein 2 (MeCP2) are highlighted. Methyl- Binding Domain (MBD) highlighted here, is responsible for the protein-DNA binding Transcription –Repression Domain (TRD) highlighted here is responsible for protein-protein interactions directly involved in gene silencing (d) Schematic representation of DNA methyltransferases (DNMTs). De novo methyl-transferase A and B (DNMT3A and B, respectively) are highlighted here. The PWWP ___domain, named for a conserved Pro-Trp-Trp-Pro motif is highlighted. The Methyl-transferase catalytic domains (MTase) of DNMT3A and DNMT3B are highlighted. e Schematic representation of Nuclear inhibitor of Protein phosphatase 1 (NIPP1) protein. Embryonic Ectoderm Development ___domain (EED) binding to Polycomb Repressive Complex 2 (PRC2) is highlighted. The Krüppel associated box (KRAB) consists of the repressive boxA and boxB domains⁸² which is schematically represented. f Schematic representation of CMV promoter targeted by AAV-KRAB-MeCP2(TRD) system. CjCas9 –matching gRNA1 and gRNA2 are highlighted. SaCas9 – matching gRNA1 and gRNA2 are highlighted. PAM of CjCas9 and SaCas9 are highlighted.