Fig. 4: Structure-guided engineering of the ωRNA scaffold.
From: Evolution-guided protein design of IscB for persistent epigenome editing in vivo
a, Schematic of OrufIscB ωRNA secondary structure model. Red, stretches that were considered for deletion; orange, pseudoknot (PK); blue, guide region. b, Left: IVTT cleavage assay with WT OrufIscB and 3′ truncated or WT ωRNA. Right: indel formation of WT ωRNA versus 3′ truncated ωRNAs in HEK293FT cells. NTS, nontargeting strand. Data are presented as mean ± s.d.; n = 3 replicate transfections. c, Indel formation mediated by OrufIscB-KRK in HEK293FT cells using ωRNA scaffolds progressively truncated from the top of the guide adapter hairpin at the 5′ end. Data are presented as mean ± s.d.; n = 3 replicate transfections. d, Indel formation using ωRNAs with combined 5′ and 3′ truncations versus WT ωRNA mediated by OrufIscB-KRK in HEK293FT cells. Data are presented as mean ± s.d.; n = 3 replicate transfections. e, Indel formation mediated by NovaIscB with WT ωRNA or combined (42-nt 5′ and 17-nt 3′) truncated ωRNA on different target sites. Data are presented as mean ± s.d.; n = 3 replicate transfections. f, Schematic of OrufIscB WT ωRNA scaffold and combined truncated ωRNA scaffold. g, Normalized ωRNA abundance in HEK293FT cells 4 days after co-transfection with NovaIscB and WT ωRNAs or combined truncated ωRNAs targeting two different genes. Data are presented as mean ± s.d.; n = 3 replicate transfections.
