Engineered Cas13a with RNA Binding Domains for Attomolar Detection

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Solution Overview

Problem

Engineering Cas13 proteins with enhanced collateral activity for nucleic acid detection has been challenging due to their complex structural dynamics, despite recent progress in optimizing the Cas13 system for rapid and ultra-sensitive detection.

Innovation Solution

The Leptotrichia wadei (Lwa) Cas13a was engineered by inserting different RNA binding domains (RBDs) into a unique active site-proximal loop, resulting in variants with significantly improved collateral activity, achieving up to 58-fold enhancement in fluorescence-based assays and enabling attomolar sensitivity detection of viral RNA targets without target preamplification.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If Cas13 proteins are engineered to enhance collateral activity for nucleic acid detection, then detection sensitivity is improved, but structural complexity and difficulty of engineering increase

Engineering Contradiction:

Improvedetection sensitivityVSAvoidstructural complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent applies local quality by inserting RNA binding domains specifically into the HEPN1 domain of Cas13a, which is proximal to the active site. This localized modification enhances RNA substrate binding affinity and collateral activity without altering the entire protein structure, thereby improving detection sensitivity while managing structural complexity.

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent employs parameter changes by modifying the Cas13a protein structure through insertion of different RNA binding domains (DRBM3, RRM1, RRM2, RRM, ZnF, Zα, Zβ) to alter its functional properties. These structural parameter changes result in enhanced collateral activity and detection sensitivity, achieving up to 58-fold enhancement in fluorescence-based assays.

Inventive Principle:

Principle #35Parameter changes

2Measurement precision

If RNA binding domains are inserted into Cas13a to improve collateral activity, then detection sensitivity increases, but protein engineering difficulty increases

Engineering Contradiction:

Improvedetection sensitivityVSAvoidprotein engineering difficulty

Core Design Contradiction:

Measurement precisionVSEase of manufacture

Solution Approach 1:

The patent applies preliminary action by identifying and characterizing the HEPN1 domain as a suitable insertion site before performing the actual domain insertion. Structural analysis of LwaCas13a was conducted first to guide the engineering strategy, allowing for targeted modifications that enhance collateral activity while simplifying the overall engineering process.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

By focusing modifications on the specific HEPN1 domain rather than the entire Cas13a protein, the patent reduces engineering complexity. The localized insertion of RNA binding domains into this specific region allows for systematic optimization of collateral activity without requiring comprehensive restructuring of the protein.

Inventive Principle:

Principle #3Local quality

3Ease of operation

If wild-type LwaCas13a is used for detection, then simplicity is maintained, but detection sensitivity is limited to picomolar levels

Engineering Contradiction:

ImprovesimplicityVSAvoiddetection sensitivity

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The patent creates composite proteins by fusing RNA binding domains with the LwaCas13a backbone. The engineered variants (RBD #3L and RBD #4L) combine the catalytic activity of Cas13a with enhanced RNA binding capabilities of the inserted domains, achieving attomolar sensitivity while maintaining operational simplicity in detection assays.

Inventive Principle:

Principle #40Composite materials

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 engineered Cas13a variants detected attomolar levels of viral RNA targets, improving the detection limit by more than 50,000 folds compared to wild-type, making it one of the most sensitive diagnostic platforms for nucleic acid detection.

Implementation Method 1

the catalytic pocket localized on the outer surface of the target-activated Cas13 complex can non-specifically cleave any surrounding RNA molecules in a characteristic 'collateral effect'

Methodology Applied

Scientific EffectCollateral effect:

Implementation Method 2

a spacer sequence that base pairs with the target RNA

Methodology Applied

Scientific EffectBase pairing:

Implementation Method 3

The resulting Cas13:crRNA:target ternary complex undergoes a large-scale conformational change in which two higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domains move toward each other to form a single catalytic pocket

Methodology Applied

Scientific EffectConformational change:

Data Source

PatentUS20250019680A1Engineered cas13 for ultrasensitive nucleic acid detection

Publication Date: 2025.01.16 WILLIAM MARCH RICE UNIVERSITY

AI summary

Provided herein are engineered Cas13 proteins with enhanced collateral activity. The engineered proteins comprise RNA binding domains (RBDs) fusions within an active site-proximal loop within a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain. Also provided are compositions comprising and methods of using the engineered Cas13 proteins to detect target nucleic acids at attomolar sensitivity.