Single-Cell TCR Cloning by CDR3 Substitution and Gibson Assembly
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Solution Overview
Problem
Current methods for cloning and expressing T-cell receptors (TCRs) are labor-intensive, time-consuming, and inefficient, particularly when dealing with small sample sizes or requiring pairing of TCR chains from single cells, and there is a lack of effective systems for γδ T cells in therapeutic applications.
Innovation Solution
A method involving RT-PCR with specific primers to obtain paired αβ or γδ TCR CDR3 sequences, followed by sequencing and cloning into a library, combined with Gibson Assembly cloning of synthesized DNA fragments into an expression vector, allowing rapid construction of TCR libraries in as little as five days.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional cloning methods are used for TCRs, then cloning can be performed with standard protocols, but the process becomes labor-intensive and time-consuming
Solution Approach 1:
The TCR cloning process is divided into distinct segments: single cell isolation, PCR amplification of TCR chains, sequencing, and assembly. This segmentation allows each step to be optimized independently and enables high-throughput processing of multiple samples simultaneously, dramatically improving productivity while reducing overall cloning time.
Solution Approach 2:
The method performs preliminary actions by pre-isolating single T cells and pre-amplifying TCR chains before final assembly. Single cells are isolated and TCR alpha and beta chains are amplified in advance, then stored for later sequencing and assembly. This preliminary preparation eliminates time-consuming steps during the final cloning process.
2Quantity of substance
If bulk sorted cells are used for TCR cloning, then large cell inputs can be processed, but pairing of TCR chains requires algorithmic imputation which fails for cells expressing two distinct TCR chains
Solution Approach 1:
Each single T cell serves itself by providing both TCR alpha and beta chains from the same cell through separate PCR amplifications. The method uses cell-specific barcodes or unique molecular identifiers that are incorporated during PCR amplification, allowing automatic and accurate pairing of TCR chains without requiring algorithmic imputation. This self-service approach ensures precise pairing even when cells express multiple TCR specificities.
3Quantity of substance
If single cell approaches are used for TCR cloning, then small sample sizes can be utilized, but efficiency decreases when dealing with high cell number inputs
Solution Approach 1:
The cloning system is designed to be universal and multi-functional, capable of handling any input size from single cells to large cell populations. The same PCR-based amplification and sequencing platform works whether processing one cell or thousands of cells simultaneously. The system can switch between single-cell mode for rare cell isolation and high-throughput mode for large cell inputs, making it adaptable to diverse experimental requirements.
4Ease of manufacture
If conventional TCR cloning methods are used, then standard protocols can be applied, but the process requires significant manual labor and optimization
Solution Approach 1:
The method replaces manual mechanical operations with automated molecular biology techniques. PCR amplification, sequencing, and bioinformatic assembly automate the previously manual steps of TCR isolation and characterization. The system substitutes hands-on manipulation with standardized molecular protocols and computational analysis, reducing manual labor while maintaining simplicity.
Data Source
AI summary
The invention provides a method for rapid cloning of T-cell receptors (TCRs) (e.g., paired αβ and γδ TCR chains) and B-cell receptors (BCRs) (e.g., paired IgH or IgK or Igλ) from single cells by CDR3 substitution using single cell PCR products and Gibson Assembly techniques and a pre-generated TCR (or BCR) library in an expression vector.