Figure 4. Evaluation of strategies to rescue ncRNA expression.

(A) Diagrams of retron editor ncRNA designs incorporating stabilizing elements. Top: circRNA design using Twister ribozyme sequences and tRNA stem forming ligation sequences. Middle: polyA RNA design incorporating a polyA AATAAA sequence before the RNAP III termination sequence. Bottom: ncRNA designs with a protective pseudoknot on the 5’ end before the retron template. (B) Northern blots of retron editor ncRNAs incorporating stabilizing elements. Radiolabeled probe targeted the scaffold sequence of the sgRNA. (C) Proposed mechanism of xrRNA-mediated RNA stability. Unprotected editor ncRNAs are susceptible to nuclease-driven decay while an xrRNA pseudoknot on the 5’ end of the ncRNA blocks degradation by cellular nucleases. (D) GFP editing outcomes for ncRNA constructs incorporating stability-enhancing elements. Error bars denote standard error of the mean.