CRISPR-Cas Recording of Cellular History via DNA Modifications
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Solution Overview
Problem
Current single-cell genomic approaches, such as single-cell RNA-seq, provide only a snapshot of cellular state and lack temporal information, making it difficult to understand how cells arrive at a specific state or endpoint.
Innovation Solution
Adaptation of the CRISPR/Cas system to record cellular events by using guide RNAs that target specific DNA sequences, allowing for the detection of gene expression and cellular history through DNA modifications, enabling the capture of temporal information on gene expression and cellular events.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-cell RNA-seq is used to profile cellular state, then detailed molecular profiling is achieved, but temporal information and cellular history are lost
Solution Approach 1:
The genome is divided into multiple target loci, each targeted by a specific guide RNA. The CRISPR-Cas system processes each locus independently, creating discrete recording events that can be separately detected and analyzed, thereby preserving temporal information while maintaining molecular profiling precision
Solution Approach 2:
The patent introduces an intermediary recording system using CRISPR-Cas mediated DNA modifications at specific genomic loci. This intermediary layer captures temporal information about cellular events and gene expression patterns, which can then be read out without disrupting the original cellular state analysis
2Adaptability or versatility
If CRISPR-Cas system is used to modify genomic content for phenotype testing, then gene function analysis is achieved, but the system cannot record cellular history
Solution Approach 1:
The CRISPR-Cas system is adapted to perform multiple functions: it can still modify genomic content for gene knockout studies while simultaneously recording cellular events through DNA modifications at recording loci. This multi-functionality allows both gene function analysis and cellular history recording to occur within the same system
Solution Approach 2:
The system uses the cell's own CRISPR-Cas machinery to record its own history. When cellular events occur that activate specific promoters, the endogenous CRISPR-Cas system automatically performs DNA modifications at recording loci, creating a self-documenting cellular history without external intervention
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Enables the recording and analysis of cellular history and events, providing a more comprehensive understanding of cellular trajectories and the factors influencing cellular states by detecting past gene expression and environmental exposures.
Implementation Method 1
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) Cas9-mediated gene disruption has been widely used in generating loss-of-function mutations in diverse organisms including mammals
Data Source
AI summary
The present invention relates to systems and methods for recording and assaying cellular events, in particular gene expression. The invention provides hereto a method of determining a cellular event of interest in a cell comprising providing a cell comprising a CRISPR-Cas system, wherein the CRISPR-Cas system comprises a guide RNA that targets a selected DNA sequence and a Cas protein capable of modifying the selected DNA sequence; whereby a nucleic acid molecule encoding at least one of the guide RNA or Cas protein is operably connected in the cell with a regulatory element comprising a promoter responsive to the cellular event, and whereby expression of at least one CRISPR-Cas system component is driven by the promoter; and determining cellular event of interest based on detection of the modification of the selected DNA sequence.