RNA-Guided FokI Nucleases for Specific Genome Editing
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Solution Overview
Problem
CRISPR-Cas RNA-guided nucleases exhibit significant off-target mutagenic effects, making it challenging to predict and minimize unwanted genomic alterations, particularly in human cells where high-frequency off-target mutations can confound research and therapeutic applications.
Innovation Solution
The development of RNA-guided FokI Nucleases (RFNs) that require dimerization for activity, specifically FokI-dCas9 fusion proteins, which use two guide RNAs to target a double-length sequence, reducing off-target mutations by ensuring precise genome editing through stringent dimerization requirements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas RNA-guided nucleases are used for genome editing, then genome editing efficiency is improved, but off-target mutagenic effects increase
Solution Approach 1:
The nuclease function is divided into two separate components: a catalytically inactive Cas9 (dCas9) that binds to the target DNA and a separate FokI nuclease domain that performs the cleavage. Two separate guide RNAs are used to recruit two FokI-dCas9 fusion proteins to adjacent target sites, and only when both are bound in close proximity does the FokI domain dimerize and cleave the DNA. This segmentation ensures that cleavage only occurs at the intended target site with high precision, eliminating off-target mutations while maintaining editing efficiency.
2Manufacturing precision
If specificity of genome editing is increased through dimerization requirements, then off-target mutations are reduced, but device complexity increases
Solution Approach 1:
The system merges the DNA-binding function of Cas9 with the cleavage function of FokI into a single fusion protein (FokI-dCas9). This merging simplifies the overall system architecture by eliminating the need for separate binding and cleavage components, while the dimerization requirement of FokI inherently provides the specificity control. The fusion protein design maintains relative simplicity while achieving high editing specificity through the biochemical properties of FokI dimerization.
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
RFNs achieve robust and highly specific genome editing in human cells, significantly reducing off-target mutations to undetectable levels, as demonstrated by deep sequencing, and provide a precise platform for research and therapeutic applications.
Implementation Method 1
The Cas9 nuclease from S. pyogenes can be guided via base pair complementarity between the first 20 nucleotides of an engineered gRNA and the complementary strand of a target genomic DNA sequence of interest
Implementation Method 2
FokI cuts each strand resulting in a pair of nicks on opposite DNA strands, thereby creating a double-stranded break
Data Source
AI summary
Many studies have shown that CRISPR-Cas nucleases can tolerate up to five mismatches and still cleave; it is hard to predict the effects of any given single or combination of mismatches on activity. Taken together, these nucleases can show significant off-target effects but it can be challenging to predict these sites. Described herein are methods for increasing the specificity of genome editing using the CRISPR/Cas system, e.g., using RNA-guided Foki Nucleases (RFNs), e.g., Fokl-Cas9 or Foki-dCas9-based fusion proteins.