Extended Data Fig. 4: Experimental validation and screening of REC grafts into OrufIscB, related to Fig. 2.
From: Evolution-guided protein design of IscB for persistent epigenome editing in vivo
(A) IVTT cleavage assay of OrufIscB (WT) with various REC insertions using IVTT protein and WT ωRNA with labeled target (target strand: Cy3; non-target strand: Cy5). Gel was imaged in Cy5 channel only. WT: Wildtype, NTS: non-target strand. (B) Indel formation mediated by WT OrufIscB with various REC insertions for two different guides in HEK293FT cells. Data are presented as mean +/- SD, n = 4 replicate transfections. (C) Schematic of the IVTT REC screening assay that tests for cleavage with varying levels of TAM distal mismatches. (D) In vitro cleavage activity score of all the screened REC domains in IVTT REC screening assay. (E) Indel formation mediated by WT OrufIscB and variants with various REC domain insertions in HEK293FT cells. Data are presented as mean +/- SD, n = 2 replicate transfections. (F) Indel formation mediated by OrufIscB (WT) vs OrufIscB-REC (OrufIscB + Nba-1 REC) across a larger panel of guides in HEK293FT cells. Data are presented as mean +/- SD, n = 4 replicate transfections.
