Extended Data Fig. 5: Point mutation selection and testing for increasing OrufIscB-REC activity, related to Fig. 2.

(A) Multiple sequence alignment and weblogo of all tested IscBs and early Cas9 (II-D) against a reference sequence of OrufIscB-REC, including the proposed point mutations for increasing activity of IscB. Most proposed point mutations were selected from the natural amino acid distribution at each position, prioritizing mutations that may improve contacts with the DNA or improve packing of the protein. The first 4 non-OrufIscB-REC sequences are shown as examples in the figure, but the multiple sequence alignment used for this analysis contains all sequences. (B) Indel formation mediated by OrufIscB-REC with each point mutation across 2 target sites. Data are presented as mean +/- SD, n = 4 replicate transfections. (C) Indel formation mediated by OrufIscB-REC with single, double, and triple mutation combinations using the top 3 mutations from distant parts of the protein that were hypothesized to combine synergistically across a panel of 12 guides. Data are presented as mean +/- SD, n = 4 replicate transfections. (D) Indel formation mediated by OrufIscB-KRK using guide lengths ranging from 12 to 28 nt at two target sites. Data are presented as mean +/- SD, n = 3. (E) Specificity analysis based on the average indel fold changes of OrufIscB-REC or OrufIscB-KRK vs WT OrufIscB and % off-target reads in total from tagmentation-based tag insertion site sequencing (TTISS) using a pool of 20 nt guides with WT OrufIscB, OrufIscB-REC, and OrufIscB-KRK.