Small Cas9 Protein Segmentation for Gene Editing Specificity
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
There is a pressing need for alternative and robust systems for targeting nucleic acids that offer improved specificity and reduced off-target effects, as existing CRISPR-Cas systems face challenges in efficiently modifying genes without causing unintended mutations.
Innovation Solution
The development of a non-naturally occurring CRISPR-Cas system comprising a Cas protein with a RuvC domain and a HNH domain, less than 850 amino acids in size, paired with a guide sequence that forms a complex to bind specifically to target sequences, and optionally includes nucleotide deaminase or transcriptional activation/repression domains to facilitate precise gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing CRISPR-Cas systems are used for gene modification, then gene editing capability is achieved, but off-target effects and unintended mutations increase
Solution Approach 1:
The Cas9 protein is divided into two separate components: a catalytically inactive Cas9 (dead Cas9) that provides targeting specificity, and a separate nucleotide deaminase domain that performs the editing function. This segmentation allows the targeting and editing functions to be optimized independently, reducing off-target effects while maintaining editing capability.
Solution Approach 2:
A guide RNA molecule serves as an intermediary between the dead Cas9 protein and the target DNA sequence. The guide RNA provides high specificity for target recognition, acting as a mediator that ensures the editing complex only binds to the intended genomic location, thereby reducing off-target effects.
2Reliability
If larger Cas proteins are used to achieve robust gene targeting, then targeting robustness is improved, but delivery efficiency decreases
Solution Approach 1:
The system segments the editing function into a smaller dead Cas9 protein combined with a separate deaminase domain, reducing the overall size of the delivery complex. This segmentation enables more efficient delivery via viral vectors or other delivery mechanisms while maintaining robust targeting through the guide RNA-directed dead Cas9 component.
Solution Approach 2:
The dead Cas9 protein is engineered with specific local properties: it retains the DNA-binding capability and PAM recognition of full-length Cas9 for robust targeting, but has the catalytic domains removed to reduce size and prevent unwanted cleavage. This local modification optimizes both targeting robustness and delivery efficiency.
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
This system enables precise modification of target sequences, reducing off-target effects and allowing for the correction of genetic mutations associated with diseases such as cancer and hemophilia, while minimizing toxicity and improving delivery efficiency.
Implementation Method 1
a guide sequence capable of forming a complex with the Cas protein and directing the complex to bind to a target sequence
Data Source
AI summary
The present disclosure provides for systems, methods, and compositions for targeting nucleic acids. In particular, the invention provides small Cas proteins and their use in modifying target sequences. In one aspect, the present disclosure provides a non-naturally occurring or engineered system comprising: a Cas protein that comprises a RuvC domain and a HNH domain, and is less than 850 amino acids in size; and a guide sequence capable of forming a complex with the Cas protein and directing the complex to bind to a target sequence.