Small Type V Endonuclease for AAV-Compatible Gene Editing
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Solution Overview
Problem
Existing CRISPR-Cas systems face challenges such as large size, limited activity in eukaryotic cells, off-target effects, immune response, complex PAM requirements, and size constraints for viral delivery, which hinder their use in gene therapy and other applications.
Innovation Solution
Development of a novel Type V CRISPR Cas nuclease, B-GEn.16, with a small size and favorable PAM sequence ('CCN') that is suitable for integration into viral vectors like AAVs, exhibiting high activity in mammalian cells and reducing off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If large Cas9 proteins from S. pyogenes are used for genome editing, then DNA cleavage activity is achieved, but the size exceeds the cargo capacity of AAV vectors
Solution Approach 1:
The invention extracts and utilizes only the essential catalytic domains (HNH and RuvC) from the full Cas9 protein, creating a minimized nuclease that retains DNA cleavage function while removing non-essential regions to reduce size for AAV delivery
Solution Approach 2:
The patent employs a transient expression strategy where the minimized Cas9 nuclease is expressed temporarily in mammalian cells to perform genome editing, then degraded, allowing repeated use of the same AAV vector system without permanent viral genome modification
2Measurement precision
If CRISPR-Cas systems are used for genome editing, then precise targeting is achieved, but off-target effects occur at similar sequences
Solution Approach 1:
The invention introduces a PAM recognition requirement adjacent to the target sequence, adding a local specificity checkpoint that distinguishes true targets from off-target sites with similar sequences but different PAM contexts
Solution Approach 2:
The minimized Cas9 nuclease is designed to require both perfect guide RNA-complementary DNA pairing and a specific PAM sequence for activation, creating a threshold effect where partial matches without correct PAM do not trigger cleavage
3Reliability
If complex PAM sequences are required for CRISPR-Cas activity, then specificity is improved, but the number of available target sites decreases
Solution Approach 1:
The invention modifies the PAM recognition parameters by engineering the minimized Cas9 to accept a simplified PAM sequence motif, changing the specificity requirements to allow broader target site availability while maintaining sufficient discrimination against off-targets
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
B-GEn.16 enables precise gene editing and manipulation in mammalian cells with reduced off-target activity, facilitating its use in therapeutic applications by overcoming size and specificity issues of existing CRISPR-Cas systems.
Implementation Method 1
an RNA guide element that is responsible to targeting these protein activities to a specific sequence on the phage DNA or RNA
Implementation Method 2
B-GEn.16, a small Type V CRISPR-Cas nuclease... Cas9 is a multi-domain enzyme that uses an HNH nuclease domain to cleave the target strand
Data Source
AI summary
Described herein are novel systems for targeting, editing or manipulating DNA in a cell or cell free environment, using novel type V B-GEn.16 (SEQ ID NO: 1) and variants thereof, as well as methods and kits for manipulating DNA.