CasM Protein RNA Cleavage via Segmented Domains
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Solution Overview
Problem
There is a need to discover and characterize new CRISPR-associated (Cas) proteins for site-specific nucleic acid engineering, as existing CRISPR-Cas systems have limitations in their applications and potential uses.
Innovation Solution
The discovery of a new Cas protein, termed CasM, which can produce single-strand breaks at RNA target sites when guided by a cognate nucleic acid guide, such as crRNA, and its use in complexes for site-specific nucleic acid engineering, including binding and cleavage of RNA targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If existing CRISPR-Cas systems are used for nucleic acid engineering, then established mechanisms are available, but application limitations and lack of versatility occur
Solution Approach 1:
The CasM protein is divided into distinct functional domains including HEPN domains for RNA cleavage, a PAM recognition domain, and a crRNA binding domain. This segmentation allows independent optimization of each function and enables modular assembly with different guide RNAs for diverse targets, resolving the contradiction between versatility and complexity.
Solution Approach 2:
CasM is designed as a universal platform that can target both DNA and RNA sequences through different guide RNA configurations. The protein maintains core functions (PAM recognition, guide binding, cleavage) while adapting to various target types, achieving broad application versatility without proportionally increasing system complexity.
2Adaptability or versatility
If new Cas proteins like CasM are discovered and characterized, then novel capabilities and expanded applications are achieved, but discovery and characterization time and resources increase
Solution Approach 1:
The CasM protein was engineered with predetermined structural features including specific HEPN domain configurations and PAM recognition motifs based on computational modeling and homology analysis. This preliminary design approach accelerated characterization by focusing experimental validation on key functional elements rather than exhaustive screening, reducing discovery time while maintaining novel capabilities.
Solution Approach 2:
Systematic variation of CasM protein parameters (amino acid substitutions in HEPN domains, guide RNA length, PAM sequence requirements) was performed to rapidly map functional boundaries. This parameter optimization strategy enabled efficient characterization of novel capabilities by identifying critical thresholds rather than requiring complete systematic analysis of all possible variations.
3Manufacturing precision
If CasM produces single-strand breaks at RNA target sites, then precise site-specific editing is achieved, but off-target effects and non-specific endonuclease activity may occur
Solution Approach 1:
The CasM protein exhibits different catalytic activities at different locations: high-fidelity PAM-dependent cleavage at the intended target site versus controlled non-specific endonuclease activity elsewhere. This spatial differentiation of function allows precise on-target editing while limiting off-target effects through regulated activation of non-specific activity only under specific conditions.
Solution Approach 2:
The crRNA guide acts as an intermediary that mediates between CasM and target sequences. It provides sequence-specific recognition that directs CasM to the correct target, reducing off-target effects. The guide RNA also regulates activation of non-specific endonuclease activity, ensuring it occurs only after proper target recognition, thus maintaining precision while enabling beneficial non-specific activity.
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
CasM enables precise and efficient site-specific nucleic acid engineering, allowing for the modification of transcription or translation of target loci, and can be used for screening and killing cells, as well as editing RNA transcripts.
Implementation Method 1
the crRNA/CasM complex is capable of binding to a first RNA target sequence complementary to the crRNA spacer sequence
Implementation Method 2
binding of the crRNA/CasM protein complex results in the cleavage of a first RNA target
Implementation Method 3
after cleavage of the first RNA target sequence by the crRNA/CasM protein complex, the complex is capable of non-specific endonuclease activity toward any single-stranded RNA in a sequence independent manner
Data Source
AI summary
A new CRISPR-associated (Cas) protein, termed “CasM,” is described, as well as polynucleotides encoding the same and methods of using CasM for site-specific genome engineering. CasM proteins are capable of targeting and cleaving single-stranded RNA.