Fig. 2: Engineering the DelIscB protein to improve editing efficiency in mammalian cells.

a An alignment was made between the predicted protein structure of DelIscB and the cryo-electron microscopy (cryo-EM) structure (7UTN) of OgeuIscB-ωRNA bound to the dsDNA target, to identify the amino acids on DelIscB that correspond to the highly active mutant residues of enIscB. b Arginine was used to replace the five amino acids of DelIscB, which structurally correspond to the highly active mutations of enIscB. Data represent mean ± s.d. of three independent biological replicates. c Multiple sequence alignment of DelIscB with its 10 homologous proteins. Representative regions are displayed, and candidates for mutagenesis are highlighted in red or cyan boxes. The type of mutant is shown at the top of the box, with red indicating a mutation to arginine or lysine and cyan indicating a mutation to other amino acid types. d Substitutions of amino acid residues of DelIscB protein. Each dot represents activity for a single variant. The dashed line represents a 1.2-fold change in activity relative to the wild type. Data represent mean ± s.d. of two independent biological replicates. e The activity of a series of mutant combinations was detected with reporter sequence EGFxxFP-target1. Data represent mean ± s.d. of three independent biological replicates. f The activity of a series of mutant combinations was confirmed with another reporter sequence (EGFxxFP-target2). Data represent mean ± s.d. of three independent biological replicates. Source data are provided as a Source data file.