Bio-orthogonal chemistry-based strategy to Turn-OFF CRISPR-Cas9 activity in solution and live cells
Bhoomika Pandit, Sweta Vangaveti, Justa F Sentre, Ian McClain, Gabriele Fuchs, Maksim Royzen
TL;DR
This paper introduces a new method to control CRISPR-Cas9 gene editing using bio-orthogonal chemistry, allowing precise regulation of gene editing in live cells.
Contribution
A novel bio-orthogonal strategy using TCO suppressors to turn off CRISPR-Cas9 activity in solution and live cells is introduced.
Findings
A Tz tag on sgRNA does not disrupt CRISPR-Cas9 activity.
TCO-modified suppressors effectively reduce nuclease activity in solution and live cells.
The method was successfully applied to multiple sgRNAs and demonstrated therapeutic potential.
Abstract
The CRISPR–Cas9 system has become a widely used gene-editing tool. Here, we present a new method for small-molecule control of CRISPR-Cas9 using bio-orthogonal chemistry between tetrazine (Tz) and trans-cyclooctene (TCO). We carried out molecular modeling studies and identified a unique position on single guide RNA (sgRNA) that can be site-specifically tagged with Tz without disrupting its activity. We also synthesized a series of TCO-modified CRISPR suppressors. When exogenously added, they bind to the Tz-tagged sgRNA, perturb the system, and drastically reduce the nuclease activity. The most successful suppressor is a TCO-modified six-amino acid-long cell-penetrating peptide, which shows excellent cell permeability. We showed that our method to control CRISPR-Cas9 nuclease activity is general by applying it to three different sgRNAs. We also showed that our method works in solution,…
Genes, proteins, chemicals, diseases, species, mutations and cell lines named across the full text — each resolved to its canonical identifier and authoritative record.
Click any figure to enlarge with its caption.
Figure 1
Figure 10
Figure 11
Figure 12
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 13Peer Reviews
No public reviews on file for this paper yet. If you reviewed it on a platform where reviews are public (OpenReview, ICLR, NeurIPS, ICML), you can paste yours below so the community can read it here.
Videos
No videos yet. Explain this paper in a talk, walkthrough, or lecture? Add one.
Taxonomy
TopicsCRISPR and Genetic Engineering · RNA Interference and Gene Delivery · RNA regulation and disease