Modified IscB Proteins for RNA-Guided DNA Cleavage

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Solution Overview

Problem

Existing approaches to engineering IscB proteins for DNA modification have limitations, necessitating improved systems for efficient RNA-guided DNA cleavage.

Innovation Solution

Modified IscB proteins with truncated or repositioned PLMP domains, along with specific amino acid mutations, are developed to enhance DNA cleavage activity and editing efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If the full-length IscB protein with intact PLMP domain is used, then the protein maintains native structure, but DNA cleavage activity and editing efficiency are limited

Engineering Contradiction:

ImproveDNA cleavage activityVSAvoidprotein structure complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The IscB protein is segmented by removing the PLMP domain (amino acids 1-54) from the N-terminus, creating a truncated version (ΔPLMP IscB) that retains DNA cleavage activity while improving productivity. This segmentation separates the regulatory PLMP domain from the catalytic core, allowing the catalytic domain to function more efficiently without the steric or regulatory constraints of the full-length protein.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The PLMP domain is extracted (removed) from the IscB protein structure. This extraction eliminates potential inhibitory effects or structural constraints imposed by the PLMP domain, thereby enhancing the DNA cleavage activity of the remaining catalytic domain while reducing overall protein complexity.

Inventive Principle:

Principle #2Taking out (Extraction)

2Productivity

If the PLMP domain is retained at the N-terminus, then the protein maintains native configuration, but editing efficiency and polynucleotide binding are reduced

Engineering Contradiction:

Improveediting efficiencyVSAvoiddomain arrangement complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The PLMP domain is repositioned from the N-terminus to the C-terminus of the IscB protein, inverting its native positional arrangement. This inversion places the regulatory domain in a different spatial context that no longer interferes with the catalytic activity and polynucleotide binding of the main body, thereby improving editing efficiency while maintaining domain connectivity.

Inventive Principle:

Principle #13The other way round (Inversion)

3Reliability

If wild-type IscB protein is used, then the system is simple, but DNA cleavage activity and stability are insufficient

Engineering Contradiction:

Improveprotein stabilityVSAvoidprotein engineering complexity

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

Specific amino acid residues within the IscB protein are mutated to improve local properties such as stability and activity. These localized mutations enhance the catalytic efficiency and structural stability of key regions without requiring complete redesign of the entire protein, thus improving reliability with minimal added complexity.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

Amino acid sequence parameters are changed through site-directed mutagenesis to optimize protein stability and DNA cleavage activity. By modifying specific residues that affect folding, stability, or catalytic function, the protein's performance parameters are improved while maintaining overall structural integrity.

Inventive Principle:

Principle #35Parameter changes

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 IscB proteins demonstrate improved DNA cleavage and editing capabilities, including increased polynucleotide binding and editing activity, with enhanced stability and yield.

Implementation Method 1

The modified IscB protein and the ωRNA bind to a target polynucleotide in a ωRNA-guided manner. The IscB protein and the ωRNA may modify the target to, for example, create an indel.

Methodology Applied

Scientific EffectRNA-guided DNA cleavage:

Implementation Method 2

During CRISPR interference, the DNA substrate is validated through R-loop formation, which involves DNA unwinding and RNA/DNA heteroduplex formation

Methodology Applied

Scientific EffectDNA unwinding and RNA/DNA heteroduplex formation:

Implementation Method 3

HNH and RuvC endonucleases are used by Cas9 to cleave the target and nontarget DNA strands, respectively

Methodology Applied

Scientific EffectEndonuclease activity: Enzyme

Data Source

PatentUS20250283116A1Use of iscb in genome editing

Publication Date: 2025.09.11 CORNELL UNIVERSITY

AI summary

Provided are modified proteins that are functional in RNA-guided DNA cleavage. The proteins include modified IscBs protein that have a modification of the N-terminus or C-terminus, or both. The modifications include a truncation of a PLMP domain of the IscB protein, or a PLMP domain that is relocated to a position of the IscB protein that is not the N-terminus. The modified IscB protein can be provided as a component of a fusion protein. The modified IscB proteins are used with an ωRNA to modify a DNA substrate.