Fig. 5: Engineering chimeric introns for multisite filtered editing.

Chimeric introns were constructed by combining the P1 or P9 helices of the Tfa introns with the Tt intron, in order to generate an intron with unique 5′ and 3′ sequence. a The P1 helix of Tt was replaced with P1 from Tfa to produce CTt. CTt was further modified to P9 from Tfa or Tfb to produce CTt9a or CTt9b, respectively. CTt9c was created by altering the native sequence of bases in the Tetrahymena P9.2 in CTt while maintaining homology across the stem loop. b Expression of oGFP by WT oRiboT or variants whose genes contained Tt, CTt, CTt9a, CTt9b, or CTt9c intron at site 2. Values and error bars represent the mean and standard deviation of n = 3 biologically independent replicates. c RT-PCR was performed on rRNA to determine whether the CTt intron was completely spliced out of oRiboT. These are representative results from n = 2 independent experiments. d Expression of oGFP by WT oRiboT or variants whose genes contained introns at site 2(oRiboT-Tt1 or oRiboT-CTt2) or sites 2 and 4 simultaneously (oRiboT-CTt2Tt4). Values and error bars represent the mean and standard deviation of n = 3 biologically independent replicates. e RT-PCR was performed on rRNA to determine whether the Tt and CTt introns were completely spliced out of oRiboT when simultaneously present at sites 2 and 4, respectively. These are representative results from n = 2 independent experiments.