PAM-Less Microbial Nucleic Acid Detection Using T7-Cas12a

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Solution Overview

Problem

Conventional CRISPR-Cas12a assays require a protospacer adjacent motif (PAM) sequence for detecting double-stranded DNA targets, limiting the selection of target sequences and reducing detection efficiency in clinical diagnostics.

Innovation Solution

A nucleic acid detection assay using CRISPR-Cas12a with a T7 transcription step that converts nucleic acid targets lacking a PAM into single-stranded RNA (ssRNA) targets, enabling detection without the need for a PAM sequence, combined with recombinase polymerase amplification (RPA) and T7 transcription.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If conventional CRISPR-Cas12a assays use PAM-dependent dsDNA detection, then the assay can detect double-stranded DNA targets, but the selection of target sequences is limited and detection efficiency is reduced

Engineering Contradiction:

Improvetarget sequence selectionVSAvoiddetection efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The invention changes the fundamental detection parameter from PAM-dependent dsDNA recognition to PAM-independent ssRNA recognition. By converting the target format and recognition mechanism, the system eliminates the PAM sequence constraint entirely, enabling detection of any target sequence without the limitations of conventional Cas12a assays

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The invention introduces T7 RNA polymerase as an intermediary component that converts dsDNA amplicons into ssRNA targets. This intermediary step bridges the gap between conventional DNA amplification methods and the novel RNA-based detection system, enabling PAM-less detection while maintaining compatibility with existing amplification workflows

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If additional steps are taken to enable PAM-less detection (artificially introducing PAM, strand displacement, nuclease degradation, or target-dependent synthesis), then random sequence detection within dsDNA amplicons becomes possible, but the assay complexity and required steps increase significantly

Engineering Contradiction:

Improvetarget sequence detectionVSAvoidassay steps

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The invention extracts and eliminates the PAM sequence requirement from the detection system entirely. Instead of working around the PAM constraint through additional steps, the system removes this limitation by switching to an RNA-based detection paradigm where PAM sequences are unnecessary, thereby simplifying the overall assay workflow

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention inverts the conventional approach by instead of trying to make dsDNA detectable without PAM through complex maneuvers, it converts the detection target to ssRNA format which naturally does not require PAM sequences for Cas12a recognition and cleavage

Inventive Principle:

Principle #13The other way round (Inversion)

3Reliability

If Cas12a is used for dsDNA detection with PAM sequences, then the assay follows conventional protocols, but the detection of PAM-less sequences is not possible

Engineering Contradiction:

Improvedetection capabilityVSAvoidtarget sequence range

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The invention creates a universal detection system that can handle both PAM-containing and PAM-less targets through a unified RNA-based approach. The T7 transcription + Cas12a RNA detection platform serves multiple functions: it works with any DNA target sequence regardless of PAM presence, maintains high detection reliability, and expands the range of detectable targets to include previously undetectable PAM-less sequences

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 assay achieves enhanced specificity and sensitivity, allowing detection of PAM-less sequences with attomolar sensitivity and improved discrimination between targets and non-targets, overcoming limitations of conventional CRISPR-Cas12a assays.

Implementation Method 1

T7 transcription step that converts nucleic acid targets lacking a protospacer adjacent motif (PAM) into single stranded RNA (ssRNA) targets

Methodology Applied

Scientific EffectTranscription: Enzyme

Implementation Method 2

Cas12a displays a distinctive trans-cleavage activity documented to result in massive indiscriminate degradation of single-stranded DNA substrate targets

Methodology Applied

Scientific EffectCRISPR-Cas12a trans-cleavage: Enzyme

Implementation Method 3

recombinase polymerase amplification (RPA) and T7 transcription

Methodology Applied

Scientific EffectRecombinase polymerase amplification: Enzyme

Data Source

PatentUS20250297333A1Systems and methods for detection of microbial nucleic acids

Publication Date: 2025.09.25 UNIV OF CONNECTICUT

AI summary

This disclosure provides compositions, methods, and systems comprising a “Universal Nuclease for Identification of Virus Empowered by RNA-sensing” (UNIVERSE) assay. The compositions, methods, and systems find use in various settings including the clinical detection of pathogen nucleic acids.