Fig. 2: Evolution-guided protein engineering of OrufIscB.
From: Evolution-guided protein design of IscB for persistent epigenome editing in vivo
a, Stepwise evolution-guided engineering of OrufIscB. The AlphaFold2 structures of WT OrufIscB, OrufIscB-REC and NovaIscB are shown. b, AlphaFold2 model of the REC domain insertion site in OrufIscB overlaid with a homologous site in DbaCas9. c, In vitro cleavage activity of selected 55 REC domain grafts in OgeuIscB with guides with progressively increasing target-matching sequence. d, AlphaFold2 model of the REC region of OrufIscB-REC with swapped loops from AlphaFold2 models of other REC domains labeled in three shades of red corresponding to regions 1, 2 and 3. The AlphaFold2 model of the swap 49 loop is shown for region 1. Conserved amino acids flanking each swap region are depicted as side chains. Sequence alignment entropy is mapped onto the structure as a white-to-blue gradient, with entropies of up to 3 being shown as blue. ZF, zinc finger. Each asterisk denotes a swap site. e, Indel formation mediated by OrufIscB-REC harboring 54 double loop swaps in the REC domain with either a 20-nt ωRNA or a 14-nt ωRNA targeting HPRT1. The indel ratio (20 nt/14 nt) and indel activity with the 20-nt guide for each variant are plotted. Combination 12 (c12, red) was selected as NovaIscB. The dashed vertical line indicates the best single-swap indel ratio. Data are presented as mean ± s.d.; n = 3 replicate transfections. f, In vitro cleavage assay with WT OrufIscB, OrufIscB-REC, NovaIscB and SpCas9 using ωRNAs or guideRNAs (gRNAs) 11 nt to 20 nt long. The experiments were repeated two times independently. g, Indel formation mediated by WT OrufIscB, OrufIscB-REC and NovaIscB using guide lengths ranging from 12 nt to 28 nt at two target sites. Data are presented as mean ± s.d.; n = 3 replicate transfections. *P < 0.05; **P < 0.01; ***P < 0.001 from the two-sided t-test for OrufIscB-REC versus NovaIscB. NS, not significant. For the DYNC1H1 guide, the P values are 0.00002 (14 nt), 0.00005 (15 nt), 0.02642 (16 nt) and 0.01150 (17 nt). For the HPRT1 guide, the P values are 0.00153 (14 nt), 0.00024 (15 nt), 0.06375 (16 nt) and 0.00130 (17 nt).
