CRISPR Cas13-Based Tools to Track and Manipulate Endogenous Telomeric Repeat-Containing RNAs in Live Cells

Abstract

TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous TERRA molecules in live cells. Here, we developed a method to visualize TERRA in live cells via a combination of CRISPR Cas13 RNA labeling and SunTag technology. Single-particle tracking reveals that TERRA foci undergo anomalous diffusion in a manner that depends on the timescale and telomeric localization. Furthermore, we used a chemically-induced protein dimerization system to manipulate TERRA subcellular localization in live cells. Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.

METTL3 drives telomere targeting of TERRA lncRNA through m6A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma

“…To elucidate the importance of m 6 A modification on TERRA RNA, we specifically removed m 6 A modification from TERRA RNA using RNA-guided CRISPR/dCasRx system (40). Catalytically dead RNA CRISPR/dCasRx fused with m 6 A demethylase-FTO (dCasRx-FTO) was recruited to TERRA RNA to specifically remove m 6 A from TERRA repeats in U-2 OS cells (41) ( Figure 3F ). We verified the removal of m 6 A from TERRA RNA by performing m 6 A RIP followed by TERRA slot blot, as evident from the blot, recruiting of dCasRx-FTO to TERRA resulted in a lower enrichment of m 6 A over TERRA RNA compared to non-template control (NTC) ( Figure 3G ).…”

SUMO Promotes DNA Repair Protein Collaboration to Support Alterative Telomere Lengthening in the Absence of PML

“…Design, synthesis, and storage of the dimerizer TFH ( T MP- F luorobenzamide- H alo) were reported previously(Lackner et al, 2022). Dimerization on telomeres was performed as previously described(Xu et al, 2022). Briefly, TFH was added directly to the growth medium to a final working concentration of 100 nM.…”

“…Cells were first treated with 2 mM thymidine (T1895, Sigma-Aldrich) for 21 h, released into fresh medium for 4 h, and then treated with 15 μM CDK1i (RO-3306, cat# SML0569, Sigma-Aldrich) for 12 h. Protein dimerization with chemical dimerizers Design, synthesis, and storage of the dimerizer TFH (TMP-Fluorobenzamide-Halo) were reported previously (Lackner et al, 2022). Dimerization on telomeres was performed as previously described (Xu et al, 2022). Briefly, TFH was added directly to the growth medium to a final working concentration of 100 nM.…”

Progress of CRISPR‐based programmable RNA manipulation and detection

“…Unlike Cas9, Cas13 directly targets ssRNA and has received more attention for its potential ability to improve sensitivity and reduce background noise. The combination of SunTag technology, which applies FP‐fused scFv (single‐chain variable fragment) with 5–10 copies of repeating epitope peptide GCN4, can increase the labeling efficiency and signal intensity of Cas13‐based imaging technologies (Figure 3c[i], middle panel; M. Xu et al, 2021). Cas13a is covalently linked to multiple tandem GCN4 peptides and recruits multiple FPs coupled with scFv to target RNA sites.…”