CasX Nuclease Domain Optimization for PAM Recognition
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Solution Overview
Problem
There is a need for additional Class 2 CRISPR/Cas systems that are optimized and offer improvements over earlier generation systems for therapeutic, diagnostic, and research applications.
Innovation Solution
Variants of the CasX nuclease protein and guide nucleic acids (gNA) are developed, with modifications to domains such as the non-target strand binding (NTSB) domain, target strand loading (TSL) domain, helical I and II domains, oligonucleotide binding domain (OBD), and RuvC DNA cleavage domain, to enhance their binding and cleavage capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If additional Class 2 CRISPR/Cas systems are developed with optimized domains, then gene editing efficiency and specificity are improved, but system complexity and development requirements increase
Solution Approach 1:
The patent applies parameter changes by systematically optimizing multiple domains of the CasX protein (NTSB, TSL, helical I and II domains, OBD, and RuvC domain) through amino acid substitutions and structural modifications. These parameter changes in protein structure and composition directly improve gene editing efficiency and specificity while managing system complexity through focused domain optimization
Solution Approach 2:
The patent achieves universality by designing a multi-functional CasX system that can target diverse PAM sequences (TTC, TTT, CTC, ATC) through optimized domain configurations. The guide nucleic acid system is also designed to be universally applicable across different target sequences, reducing the need for entirely different systems for various applications
2Adaptability or versatility
If the CasX system is optimized to target a wider range of PAM sequences, then adaptability is improved, but manufacturing precision and optimization requirements increase
Solution Approach 1:
The patent applies local quality by making specific, targeted modifications to particular domains of the CasX protein (such as the PAM-interacting regions in the NTSB and TSL domains) to enable recognition of diverse PAM sequences. This localized optimization approach allows the system to achieve broad PAM compatibility while maintaining precise control over the modification requirements and avoiding genome-wide optimization complexity
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 modified CasX variants and gNA exhibit improved characteristics such as enhanced gene editing efficiency, increased specificity, and the ability to target a wider range of PAM sequences, leading to more effective genome manipulation.
Implementation Method 1
The CasX variant is capable of forming a complex with a guide nucleic acid (NA), and wherein the complex can bind a target DNA
Implementation Method 2
a RuvC DNA cleavage domain
Data Source
AI summary
Provided herein are catalytically dead engineered proteins, e.g., catalytically dead CasX proteins. Also provided herein are systems comprising guide nucleic acids and catalytically dead engineered proteins, and methods for use thereof.