CRISPR Cas9 Genome Editing for Restriction-Site-Free Mutation Detection
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Solution Overview
Problem
Existing genome editing methods using the CRISPR/Cas system have not been developed, and Restriction Fragment Length Polymorphism (RFLP) is limited by the availability of appropriate restriction sites, making it challenging to detect engineered nuclease-mediated mutations effectively.
Innovation Solution
A composition and method using a guide RNA specific for target DNA and Cas protein-encoding nucleic acid or Cas protein for targeted DNA cleavage and mutagenesis in eukaryotic cells, enabling programmable RNA-guided endonucleases (RGENs) for RFLP analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If RFLP method is used for detecting engineered nuclease-mediated mutations, then genotyping can be performed, but it is limited by the lack of appropriate restriction sites at the target site of interest
Solution Approach 1:
The patent introduces T7 endonuclease I or Surveyor nuclease as an intermediary that can recognize and cleave mismatched DNA sequences. Instead of relying on restriction sites in the target DNA, the system uses these nucleases to detect mutations created by engineered nucleases, thereby overcoming the limitation of unavailable restriction sites while maintaining mutation detection capability
Solution Approach 2:
The patent replaces the mechanical restriction enzyme recognition system with a biochemical mismatch detection system. Rather than using restriction enzymes that require specific recognition sequences, the system employs T7 endonuclease I or Surveyor nuclease that recognize structural mismatches in DNA heteroduplexes, substituting one detection mechanism for another that is more versatile
2Measurement precision
If T7 endonuclease I or Surveyor nuclease assays are used to detect engineered nuclease-mediated mutations, then mutation detection is possible, but the assays are cumbersome and tend to underestimate mutation frequencies
Solution Approach 1:
The patent modifies the detection parameters by using RFLP analysis on PCR-amplified products containing the mutation site. By changing from direct enzymatic assays to a PCR-RFLP workflow, the system improves ease of operation while maintaining accurate mutation frequency detection, as PCR amplification enriches the target sequences and RFLP provides clear binary detection of mutant versus wild-type alleles
3Measurement precision
If mutant sequences form homoduplexes with each other, then they cannot be distinguished from wildtype cells using T7 endonuclease I or Surveyor nuclease assays, but this limits the ability to detect homozygous bi-allelic mutant clones
Solution Approach 1:
The patent performs preliminary PCR amplification of the target region containing the mutation site before RFLP analysis. This preliminary action enriches the mutant sequences and creates sufficient material for clear RFLP detection, allowing distinction between homozygous mutants, heterozygous mutants, and wild-type cells based on fragment patterns, thereby improving genotype discrimination accuracy and reliability
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
The approach allows for the detection and analysis of almost any single nucleotide polymorphism or small insertion/deletion (indel) via RGEN-mediated RFLP, providing a new genome editing tool for detecting and cleaving naturally-occurring variations and mutations.
Implementation Method 1
a guide RNA specific for target DNA
Implementation Method 2
Cas protein forms an active endonuclease when complexed with two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), thereby slicing foreign genetic elements
Data Source
AI summary
The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for compositions and methods that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. For example, disclosed herein is a method of producing an engineered eukaryotic cell. In some embodiments, the method may comprise preparing a Cas9 protein, preparing a sgRNA, and preparing a cell-free buffer. The method may further comprise disposing the Cas9 protein and sgRNA in the cell-free buffer to provide for a transfection mixture and transfecting the transfection mixture into a eukaryotic cell, wherein a Cas9/sgRNA complex formed by the Cas9 protein and the sgRNA induces a modification of a target endogenous DNA sequence in the nucleus of the eukaryotic cell to provide for an engineered eukaryotic cell.