Self-targeting Genome Editing System

Applications

This technology can be used to record molecular events in living cells that happen at short timescales in a long-term, continuous, accumulative, and autonomous manner.

Problem Addressed

Molecular recording technologies allow for conversion of transient biological signals into permanent records that can be retrieved later for analysis. Current technologies are capable of recording binary on or off biological signals but are unable to capture analog signals such as duration and/or magnitude of the signal. The current technology employs targeted DNA mutagenesis using CRISPR system to boost the mutation rate without compromising cell viability, and allows for accumulation of mutations to capture information on the strength of the signal.

Technology

The technology works on the principle of recording continuous and accumulative mutations at a target site introduced by CRISPR mediated DNA breaks and subsequent error prone repairs. The power of the technology comes from using self-targeting guide RNA (stgRNA) generated by adding PAM sites to guide RNA. Error prone repair of CRISPR mediated cleavage results in changes in the sequence of stgRNA. If the PAM site is maintained, this process is repeated multiple times for continuous accumulation of mutations.

This targeted mutagenesis can be operationally linked to a biological event of interest by linking expression of stgRNA or Cas9 to the biological event. Accumulative mutations give information about duration and/or intensity of the signal. A chronological order and lineage of cell division can be established using sequencing analysis on this population of cells.

Advantages

  • High mutation rate directed towards a target site allows for long-term continuous and accumulative recording without affecting overall cell viability
  • Linkage of mutation induction to biological signal allows for recording duration and/or magnitude of the signal
  • Easy readout using sequencing of the targeted site only