1.Design Overview
Rapid and specific identification of respiratory pathogens requires molecular components that are not only sensitive, but also modular and reusable.
To support our RPA–Cas12a detection platform, we developed a modular Part collection covering the biosynthesis of the core LbCas12a nuclease and pathogen-specific assay components for target amplification and recognition.
Once the core LbCas12a nuclease is available, the detection system follows a common molecular workflow: Target Recognition → RPA Amplification → Cas12a Recognition → Signal Generation
To organize our Parts according to their roles in the overall platform, we divided the collection into two functional layers:
1.1 Core Enzyme Biosynthesis Layer
This layer provides the core LbCas12a nuclease required for the detection platform.
It includes:
E. coli B codon-optimized LbCas12a CDS
T7-driven His₆–TEV–LbCas12a Expression Cassette
1.2 Pathogen-Specific Recognition Layer
This layer determines which pathogen is detected.
It includes:
Synthetic target templates
RPA primer pairs
Cas12a crRNAs
Together, these Parts form a modular toolkit for expandable nucleic-acid detection.
2. Collection at a Glance
2.1 Core Enzyme Biosynthesis Parts
| Part | Main Role | Suggested Type | BBa # |
|---|---|---|---|
| E. coli B codon-optimized LbCas12a CDS | Core LbCas12a coding sequence | CDS / Basic Part | BBa_26806TBI |
| T7-driven His6-TEV- LbCas12a | Recombinant LbCas12a production | Engineered Region / Composite Part | BBa_26OVD7TR |
2.2 Pathogen-Specific Parts
| Part | Synthetic Target | Forward Primer | Reverse Primer | Cas12a crRNA |
|---|---|---|---|---|
| Respiratory syncytial virus | BBa_26RITQGC | BBa_26M42NDA | BBa_26IVYHX0 | BBa_26LPPRQU |
| Human metapneumovirus-B | BBa_268ZOMYX | BBa_26C5IQYJ | BBa_26Q062XR | BBa_269BNB8L |
| Haemophilus influenzae | BBa_26G8N35H | BBa_26OB5PRH | BBa_26E3BKR1 | BBa_26IXTE4N |
| Streptococcus pneumoniae | BBa_26Q36DFE | BBa_26HAUKYN | BBa_26WQYC45 | BBa_263AQFBT |
| Bordetella pertussis | BBa_260SEHAI | BBa_26H3W838 | BBa_26K5KK6T | BBa_269UE9N9 |
2.3 LbCas12a Biosynthesis Parts
LbCas12a serves as the shared core nuclease of our RPA–Cas12a detection platform. To support recombinant production of this enzyme, we designed two biosynthesis-related Parts: a codon-optimized LbCas12a CDS and a complete T7-driven expression cassette.
| Part Name | Part Type | Function |
|---|---|---|
| E. coli B codon-optimized LbCas12a CDS |
CDS / Basic Part | Encodes the LbCas12a nuclease derived from Lachnospiraceae bacterium ND2006. The CDS was codon-optimized for recombinant expression in E. coli and serves as a reusable core nuclease coding module. |
| T7-driven His6-TEV- LbCas12a Expression Cassette | Engineered Region / Composite Part |
Provides a complete T7-driven expression unit for recombinant production of LbCas12a in E. coli BL21(DE3), incorporating elements for inducible expression, His₆-mediated affinity purification, and optional TEV-mediated tag removal. |
2.4 Synthetic Target Templates
Synthetic target templates provide defined DNA substrates for assay development.
They represent selected pathogen-associated genomic regions and can serve as positive-control templates for RPA amplification and downstream Cas12a-based detection.
Using synthetic DNA allows the molecular workflow to be developed without requiring intact pathogenic organisms.
| Pathogen | Part Name | Part Type | Function |
|---|---|---|---|
| Respiratory syncytial virus (RSV) | RSV N Gene Synthetic Target Template | PCR product / Basic Part | A synthetic, non-infectious DNA target template containing a conserved region of the HRSV N gene, designed to serve as a positive control for RPA amplification and CRISPR-Cas12a-based nucleic acid detection of HRSV, including subgroup A and subgroup B. |
| Human metapneumovirus-B (hMPV-B) | hMPV-B N Gene Synthetic Target Template | PCR product / Basic Part | A synthetic, non-infectious DNA target template containing a conserved region of Human metapneumovirus-B, designed to serve as a positive control for RPA amplification and CRISPR-Cas12a-based nucleic acid detection |
| Haemophilus influenzae (HI) | HI ompP6 Gene Synthetic Target Template | PCR product / Basic Part | A synthetic, non-infectious DNA target template containing a conserved region of Haemophilus influenzae, designed to serve as a positive control for RPA amplification and CRISPR-Cas12a-based nucleic acid detection |
| Streptococcus pneumoniae | SPN lytA Gene Synthetic Target Template | PCR product / Basic Part | A 251 bp Streptococcus pneumoniae target amplicon containing the target sequence required for recognition by the SPN-specific Cas12a crRNA. It is intended to serve as the amplification target and positive-control template in an RPA-Cas12a nucleic acid detection system. |
| Bordetella pertussis | BP IS1663 Gene Synthetic Target Template | PCR product / Basic Part | A synthetic, non-infectious DNA target template containing a selected target region from Bordetella pertussis. It is designed to serve as a positive-control template for RPA isothermal amplification and subsequent CRISPR-Cas12a-based nucleic acid detection. |
2.5 RPA Primer Parts
Recombinase Polymerase Amplification enables rapid amplification of DNA under isothermal conditions.
For each pathogen, a corresponding forward and reverse primer pair forms the amplification component of the pathogen-specific layer.
| Pathogen | Primer Role | Part Name | Role in Our System |
|---|---|---|---|
| Respiratory syncytial virus (RSV) | Forward Primer | RSV N Gene RPA Forward Primer | RSV N Gene primer pair: amplifies the selected HRSV Ngene target for downstream Cas12a recognition. |
| Reverse Primer | RSV N Gene RPA Reverse Primer | ||
| Human metapneumovirus-B (hMPV-B) | Forward Primer | hMPV-B N Gene RPA Forward Primer | hMPV-B N Gene primer pair: form the pathogenspecific RPA amplification module for hMPV-B detection. |
| Reverse Primer | hMPV-B N Gene RPA Reverse Primer | ||
| Haemophilus influenzae (HI) | Forward Primer | HI ompP6 Gene RPA Forward Primer | HI ompP6 Gene primer pair: amplifies the selected H. influenzae target sequence and generates the substrate for downstream Cas12a recognition. |
| Reverse Primer | HI ompP6 Gene RPA Reverse Primer | ||
| Streptococcus pneumoniae | Forward Primer | SPN lytA Gene RPA Forward Primer | SPN lytA Gene primer pair: amplifies the selected 251 bp Streptococcus pneumoniae target sequence and generates the substrate for downstream Cas12a recognition. |
| Reverse Primer | SPN lytA Gene RPA Reverse Primer | ||
| Bordetella pertussis | Forward Primer | BP IS1663 Gene RPA Forward Primer | BP IS1663 Gene primer pair: amplifies the selected Bordetella pertussis target sequence and generates the substrate for downstream Cas12a recognition. |
| Reverse Primer | BP IS1663 Gene RPA Reverse Primer |
2.6 Cas12a crRNA Parts
RPA provides target amplification, while Cas12a crRNAs provide sequence-specific recognition.
Each crRNA contains a guide region corresponding to the target sequence.
Following recognition of the amplified DNA, the Cas12a–crRNA complex activates Cas12a-mediated collateral cleavage for downstream signal generation.
Therefore, the crRNA functions as the recognition module of our detection platform.
| Pathogen | Part Name | Role in Our System |
|---|---|---|
| Respiratory syncytial virus (RSV) | RSV N Gene-targeting LbCas12a crRNA | This synthetic crRNA contains an LbCas12acompatible directrepeat region and an HRSVspecific spacer.It guides LbCas12a toward the selected HRSV target sequence. |
| Human metapneumovirus-B (hMPV-B) | hMPV-B N Gene-targeting LbCas12a crRNA | This crRNA guides Cas12a recognition of the hMPV-B target sequence following RPA amplification. |
| Haemophilus influenzae (HI) | HI ompP6 Gene-targeting LbCas12a crRNA | This synthetic Cas12a crRNA guides recognition of the selected H. influenzae target following RPA amplification. |
| Streptococcus pneumoniae | SPN lytA Gene-targeting LbCas12a crRNA | This synthetic Cas12a crRNA contains a Cas12a-compatible direct-repeat region and an SPN-specific spacer. It guides Cas12a to recognize the complementary target sequence within the SPN RPA amplicon following RPA amplification. |
| Bordetella pertussis | BP IS1663 Gene-targeting LbCas12a crRNA | This synthetic Cas12a crRNA guides recognition of the selected Bordetella pertussis target following RPA amplification. |
Functional Classification
3.1 Biosynthesis Module
Provides the core LbCas12a nuclease required for the detection platform.
Includes:
E. coli B codon-optimized LbCas12a CDS
3.2 Target and Recognition Module
Determines molecular specificity.
Includes:
Synthetic target templates
Pathogen-specific RPA primers
Cas12a crRNAs
3.3 Amplification Module
Supports pathogen-specific nucleic-acid amplification.
Includes:
RPA primer pairs
From Parts to a Modular Detection Toolkit
Our Part collection can therefore be summarized as:
Foundation Step - Produce the Core Enzyme
Use the LbCas12a biosynthesis Parts to support recombinant production of the core LbCas12a nuclease.
Step 1 - Select the Pathogen
Choose the pathogen-specific target.
Step 2 - Amplify the Target
Use the corresponding RPA primer pair.
Step 3 - Recognize the Amplicon
Use the corresponding Cas12a crRNA together with LbCas12a to recognize the amplified target.
The key principle is simple:The pathogen-specific components change, while the core LbCas12a biosynthesis module can remain reusable across different pathogen-specific assays.