CasX Endonuclease Guide RNA Specificity for Uncultured Organism Discovery
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Solution Overview
Problem
Current CRISPR-Cas technologies are primarily based on systems from cultured bacteria, leaving untapped the vast majority of organisms that have not been isolated, and there is a need for additional Class 2 CRISPR/Cas systems, such as Cas protein plus guide RNA combinations.
Innovation Solution
The development of RNA-guided endonuclease polypeptides, referred to as CasX polypeptides, and their associated guide RNAs, which provide sequence specificity and are useful in various applications, including genome manipulation, along with archaeal Cas9 polypeptides and their guide RNAs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If CRISPR-Cas technologies are based on systems from cultured bacteria, then the technology is well-established and reliable, but the vast majority of organisms that have not been isolated remain untapped, limiting diversity and potential applications
Solution Approach 1:
The patent uses metagenomic sequencing as an intermediary tool to access CRISPR-Cas systems from uncultured organisms. By sequencing DNA directly from environmental samples rather than requiring bacterial culture, the technology bridges the gap between established bacterial-based CRISPR systems and the vast uncultured microbial diversity, enabling discovery of novel Cas proteins and guide RNA combinations from organisms that cannot be grown in laboratory conditions
Solution Approach 2:
The patent replaces the traditional mechanical/cultural approach of growing and isolating bacteria with a molecular-based metagenomic approach. Instead of physically culturing organisms to extract CRISPR systems, the method uses DNA extraction and high-throughput sequencing to directly access genetic material from environmental samples, substituting biological cultivation with biochemical and computational methods to discover novel CRISPR-Cas systems
2Adaptability or versatility
If only a few Class 2 CRISPR/Cas systems have been discovered, then the technology is simpler to implement, but there is a need for additional Class 2 CRISPR/Cas systems to expand capabilities
Solution Approach 1:
The patent employs bioinformatics pipelines that provide feedback loops for identifying and characterizing novel CRISPR-Cas systems. The metagenomic sequencing data is analyzed using computational algorithms that compare sequences against known CRISPR-Cas databases, providing feedback on system identification and classification. This feedback mechanism enables systematic discovery of new Class 2 CRISPR/Cas systems while maintaining organization and reducing complexity through automated classification
Solution Approach 2:
The patent develops universal methodologies for identifying CRISPR-Cas systems across diverse organisms. The metagenomic approach and bioinformatics pipeline are designed to be universally applicable to any environmental sample, enabling the same systematic process to discover multiple different Class 2 CRISPR/Cas systems from various sources. This universal framework expands CRISPR system variety without proportionally increasing complexity, as the same multi-functional pipeline handles diverse inputs
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
These new CRISPR systems enable programmable DNA interference and editing, offering expanded capabilities for genome manipulation and providing sequence specificity, which was previously limited by the availability of Class 2 CRISPR/Cas systems.
Implementation Method 1
RNA-guided endonuclease polypeptides, referred to herein as 'CasX' polypeptides
Implementation Method 2
guide RNAs (referred to herein as 'CasX guide RNAs') that bind to and provide sequence specificity to the CasX proteins
Implementation Method 3
RNA-guided endonuclease polypeptides, referred to herein as 'CasX' polypeptides... enable programmable DNA interference and editing
Data Source
AI summary
The present disclosure provides CasX proteins, nucleic acids encoding the CasX proteins, and modified host cells comprising the CasX proteins and/or nucleic acids encoding same. CasX proteins are useful in a variety of applications, which are provided. The present disclosure provides CasX guide RNAs that bind to and provide sequence specificity to the CasX proteins, nucleic acids encoding the CasX guide RNAs, and modified host cells comprising the CasX guide RNAs and/or nucleic acids encoding same. CasX guide RNAs are useful in a variety of applications, which are provided. The present disclosure provides archaeal Cas9 polypeptides and nucleic acids encoding same, as well as their associated archaeal Cas9 guide RNAs and nucleic acids encoding same.