Extended Data Fig. 6: Structure guided engineering of the REC domain in OrufIscB-REC, related to Fig. 2.
From: Evolution-guided protein design of IscB for persistent epigenome editing in vivo
(A) IVTT cleavage assay of OrufIscB-REC with 12 single loop swaps in the REC domain using IVTT protein and WT ωRNA with labeled target (target strand: Cy3; non-target strand: Cy5). Gel was imaged in Cy5 channel only. NTS: non-target strand. (B) Indel formation mediated by OrufIscB-REC harboring 52 single loop swaps in the REC domain with either a 20-nt ωRNA or a 14-nt ωRNA targeting HPRT1, DYNC1H1 and DYRK1A. The indel ratio (20 nt/14 nt) and indel activity with the 20-nt guide for each variant are plotted. The selected candidates for further combination tests were labeled in shades of red. Double dagger indicates swap 49. Data are presented as mean +/- SD, n = 3 replicate transfections. (C) Cryo-EM ternary complex structure of OrufIscB-REC-swap 49 with a native ωRNA and target DNA heteroduplex with a focus on the hydrophobic interactions between the engineered REC domain and the guide:target heteroduplex. Swap 49 in swap region 3 is shown in red. (D) 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 DYNC1H1. The indel ratio (20 nt/14 nt) and indel activity with the 20-nt guide for each variant are plotted. Variant c12 (red, swap combo 12) was selected as NovaIscB. Data are presented as mean +/- SD, n = 3 replicate transfections. (E) In vitro cleavage assay with OrufIscB-REC with swap combo 6 using ωRNAs 11 to 20-nts long. (F) On-target indel formation at 4 target sites mediated by WT OrufIscB, OrufIscB-REC, NovaIscB or OrufIscB-KRK in TTISS samples using 14-nt (top) or 20-nt (bottom) guide pool. (G) Specificity analysis of OrufIscB-REC, NovaIscB, OrufIscB-KRK and WT OrufIscB with 14-nt guides. % off-target reads is the average of the number of off-target reads divided by total number of reads for each guide from TTISS using a pool of four 14-nt guides. Indel fold change is the average of fold change of percent indels generated relative to WT OrufIscB across the same four guides as measured by amplicon sequencing of the same samples. Error bars represent standard deviation. The numbers of detected off-target sites are included in brackets. (H) Specificity analysis of OrufIscB-KRK with 20-nt guides. X and Y axes are calculated as in (G). (I) On-target indel formation at 4 target sites mediated by NovaIscB, enOgeuIscB, eIscB, AsCas12f-YHAM and SpCas9 in TTISS samples using individually transfected guides.
