CRISPR/Cas12a Detection of Bi-Allelic CD71 Gene-Edited Cells
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Solution Overview
Problem
Efficient and accurate detection of bi-allelic gene edited cells, particularly those with CRISPR/Cas9-induced modifications, is challenging due to complex operations and low sensitivity in existing methods.
Innovation Solution
A CRISPR/Cas12a system utilizing a crRNA that targets the CD71 gene, specifically designed to recognize and cleave the third exon of the pig CD71 gene, combined with a primer pair and DNA probes for sensitive and specific detection of bi-allelic gene edits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing detection methods are used for bi-allelic gene edited cells, then detection can be performed, but the operation becomes complex and sensitivity decreases
Solution Approach 1:
The invention changes the detection parameters by using CRISPR/Cas12a system with specifically designed crRNA that targets the CD71 gene third exon. The system uses fluorescently labeled DNA probes with specific sequences that bind to the target region, enabling sensitive detection through fluorescence signal changes rather than complex operational procedures
Solution Approach 2:
The invention introduces intermediary molecules including crRNA (clustered regularly interspaced short palindromic repeats) and DNA probes as mediators between the Cas12a enzyme and the target DNA. These intermediaries enable specific recognition and binding, allowing sensitive detection while simplifying the overall detection process through molecular mediation rather than complex operational steps
2Measurement precision
If CRISPR/Cas12a system is used with specifically designed crRNA and DNA probes, then detection sensitivity and accuracy improve, but the system complexity increases
Solution Approach 1:
The invention achieves universality by designing a modular CRISPR/Cas12a detection system where the core Cas12a enzyme can work with different crRNA and DNA probe combinations. The system uses universal components (Cas12a protein, fluorescent labels, quencher molecules) that can be adapted to detect different target sequences, reducing overall system complexity while maintaining high detection accuracy for the CD71 gene
Solution Approach 2:
The invention segments the detection system into distinct functional modules: the Cas12a enzyme module, the crRNA guide module, and the fluorescent DNA probe module. Each module performs a specific function (cleavage, recognition, signal generation) and can be optimized independently. This segmentation allows high detection accuracy through specialized molecular components while managing system complexity through functional separation
3Productivity
If traditional detection methods are used, then the procedure is established, but the detection process is time-consuming
Solution Approach 1:
The invention replaces traditional mechanical or manual detection methods with a biochemical amplification system based on CRISPR/Cas12a. The system uses enzymatic cleavage of DNA probes by Cas12a to generate fluorescent signals, eliminating time-consuming manual operations and enabling rapid automated detection of bi-allelic gene edited cells
Solution Approach 2:
The invention implements continuous useful action through the amplification mechanism of the CRISPR/Cas12a system. Once the crRNA-Cas12a complex binds to the target DNA, it continuously cleaves fluorescent probes, generating a cumulative fluorescent signal that increases over time. This continuous signal amplification enables rapid detection without repeated sampling or processing steps, significantly reducing detection time
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 system enables rapid, accurate, and cost-effective screening of bi-allelic gene edited cells, demonstrating high sensitivity and specificity in identifying CD71 gene edits, even at low concentrations.
Implementation Method 1
CRISPR/Cas12a system is a ribonucleic acid (RNA) guided DNA endonuclease-based technology, the core of which lies in formation of a complex between Cas12a protein and specifically designed CRISPR RNA (crRNA). This complex can accurately recognize and target DNA regions containing specific PAM sequences, and perform cleavage downstream the PAM sequence to produce DNA double strand breaks.
Implementation Method 2
After cleaving a target DNA, Cas12a protein enters an activated state and exhibits non-specific incidental cleavage activity, which can cleave nearby non target DNA molecules.
Implementation Method 3
the single stranded DNA probe is labeled with different groups at both ends; and/or the groups are fluorescent groups, quenching groups, and/or a biotin
Data Source
AI summary
A method for detecting bi-allelic gene edited cells based on CRISPR/Cas12a technology is provided. The method provides crRNA, a nucleotide sequence of which that binds target is SEQ ID NO: 6. The crRNA is transcribed from a transcription template in vitro; the transcription template is a product obtained by annealing a single stranded DNA molecule shown in SEQ ID NO: 10 and a single stranded DNA molecule shown in SEQ ID NO: 13. The method provides crRNA of CD71 gene and constructs a CRISPR/Cas12a system using the same for CRISPR/Cas12a detection, achieving the screening for CRISPR/Cas9 induced CD71 gene bi-allelic gene edited cells. This method is simple, easy, fast, and cost-effective for screening a large number of mutant cells.