Skip to main content
[Preprint]. 2024 Feb 1:2024.01.31.578223. [Version 1] doi: 10.1101/2024.01.31.578223

Figure 4 |. Evaluation of protein level edits, transcriptome-wide specificity, and delivery by lentivirus.

Figure 4 |

(a) Evaluation of USF1 protein editing due to PRECISE-based RNA writing by Western Blotting. USF1 is overexpressed and is targeted with either targeting or non-targeting DisCas7-11 guides and targeting or non-targeting cargo hybridization guides. Shown is the RNA editing rate (top) and Western blot gel with semi-quantitative protein editing rate (bottom). Arrows indicate either the unedited protein (blue) or the edited trans-spliced protein (black). (b) Evaluation of endogenous HDAC1 protein editing due to PRECISE-based RNA editing by Western Blotting. The HDAC1 transcript is targeted with either targeting or non-targeting DisCas7-11 guides and targeting or non-targeting cargo hybridization guides. Shown is the RNA editing rate (top) and Western blot gel with semi-quantitative protein editing rate (bottom). Arrows indicate either the unedited protein (blue) or the edited trans-spliced protein (black). (c) Evaluation of endogenous PPIB protein editing due to PRECISE-based RNA writing by Western Blotting. The PPIB transcript is targeted with either targeting or non-targeting DisCas7-11 guides and a targeting cargo hybridization guide. Shown is the RNA editing rate (top) and Western blot gel with semi-quantitative protein editing rate (bottom). Arrows indicate either the unedited protein (blue) or the edited trans-spliced protein (black). (d) Volcano plots of differentially expressed genes as measured by transcriptome-wide sequencing of cells with PPIB PRECISE editing. (e) Number of significant trans-spliced off-targets identified by transcriptome-wide sequencing of cells following PRECISE editing of PPIB. (f) Schematic of PRECISE RNA writing for 3′ trans-splicing via lentiviral delivery. Shown are the construct designs for a two-vector system. (g) Demonstration of PRECISE editing of the SHANK3 transcript with lentiviral delivery of DisCas7-11 expression, guides, and RNA trans-template in HEK293FT cells. RNA writing of a 108 bp sequence into the endogenous SHANK3 transcript with targeting and non-targeting DisCas7-11 guides is compared.