Fig. 1: Filtered editing: methods to edit and diversify repetitive genetic elements.
From: Targeted editing and evolution of engineered ribosomes in vivo by filtered editing
a There are seven native ribosomal operons (light blue) in the E. coli genome, which share extensive sequence homology to an orthogonal-tethered ribosome (oRiboT, dark blue), which is also introduced into the genome. b The secondary structure of oRiboT rRNA. Introns were introduced at four separate sites in oRiboT. Intron insertion sites are designated with purple arrows, and areas that can be targeted with filtered editing highlighted in purple. c, d In f-CRISPR and f-MAGE, an intron is introduced near the site targeted for mutagenesis to provide a unique address for hybridization of c sgRNA to introduce a Cas9-mediated double-stranded break, or d a pool of mutagenic MAGE ssODNs. After the mutation is introduced into the DNA, the intron is spliced out of the transcribed RNA to produce the desired product. In this work the Tetrahymena thermophila and a panel of other group 1 self-splicing introns were introduced into the gene encoding the orthogonal-tethered ribosome oRiboT to distinguish it from the seven native ribosome genes; f-CRISPR and f-MAGE were then used to generate libraries of spliced oRiboT RNAs with targeted mutations.
