Extended Data Fig. 6: Design and screen of sesRNAs targeting Fezf2 and Ctip2 RNAs in vitro and in vivo.
From: Programmable RNA sensing for cell monitoring and manipulation
a–b, Genomic structures of mouse Fezf2 (a) and Ctip2 (b) genes with locations of various sesRNAs as indicated. c, List of sesRNAs and Fezf2 and Ctip2 target gene fragments used for sesRNA screen.d, In Target vectors CAG-BFP-Fezf2 or CAG-BFP-Ctip2, a 200–3000 bp genomic region of the Fezf2 or Ctip2 gene containing sequences complementary to a sesRNA in a, b were cloned downstream to the BFP and T2a coding region driven by a CAG promoter. In READR vectors, READRFezf2-GFP or READRCtip2-GFP expresses corresponding sesRNAs shown in a, b. e–f, Quantification of efficiencies READRFezf2-GFP (e) or READRCtip2-GFP (f) as GFP conversion ratio by FACS assay of 293T cells co-transfected with CAG-BFP-Fezf2 or CAG-BFP-Ctip2 target vector, respectively. g, Schematic of binary READR AAV vectors. In READR vector, a hSyn promoter drives expression of mCherry followed by sequences coding for sesRNACtip2, smFlag and tTA2 effectors. In Reporter vector, TRE3g promoter drives mNeon in response to tTA2 from the READR vector. h, Coronal sections of mouse cortex injected with binary READRFezf2 vectors. mNeon indicated READRFezf2 labeled cells. Four Fezf2 sesRNAs were screened. i, Quantification of specificity of 4 Fezf2 sesRNAs in h. For Fezf2 sesRNA in-vivo screen, the specificity of each sesRNA was calculated by co-labeling by READR AAVs and CTIP2 antibody (due to lack of FEZF2 antibody); as Ctip2 represents a subset of Fezf2+ cells (not shown), CTIP2 antibody gives an underestimate of the specificity of Fezf2 sesRNA. SesRNA1 showed highest specificity. j, Coronal sections of mouse cortex injected with binary READRCtip2 vectors. mNeon indicated binary READR labeled cells. Eight Ctip2 sesRNAs were screened. k, Quantification of specificity of 8 sesRNAs in j. The specificity of each sesRNA was calculated by co-labeling by binary READRCtip2 AAVs and CTIP2 antibody (not shown). SesRNA3 and sesRNA8 showed highest specificity. Error bars in e–f are mean values ± s.e.m. n = 3, n represents the number of independent experiments performed. Bars in i and k are values from one mouse performed. Each bar in i and k is the value from one mouse performed (n = 1).
