cliCRISPR: crRNA-limited CRISPR/Cas12a system for multiplexed detection

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR/Cas12a is widely used for nucleic acid detection, typically requiring excess crRNA.
• Non-specific trans-cleavage activity of CRISPR/Cas12a hinders multiplexed detection in single-pot assays.
• Existing methods face challenges in achieving sensitive and specific multiplexed detection.

Purpose of the Study:

• To introduce a crRNA-limited strategy, cliCRISPR, for multiplexed detection using the CRISPR/Cas12a system.
• To demonstrate that fluorescence intensity can be correlated with crRNA concentration for multiplexed quantification.
• To validate the cliCRISPR method for precise genotyping of genetic variations.

Main Methods:

• Developed a crRNA-limited CRISPR/Cas12a strategy (cliCRISPR).
• Correlated fluorescence intensity with specific crRNA concentrations for distinct signal levels.
• Applied a logic-gate strategy for genotyping rs4646536, a variant linked to vitamin D deficiency.
• Validated the method using clinical samples and compared results with TaqMan qPCR.

Main Results:

• Achieved distinguishable fluorescence intensities using controlled crRNA concentrations (10 nM crRNA1, 3 nM crRNA2).
• Successfully genotyped rs4646536 (wild-type, mutant, heterozygous) with high accuracy.
• Demonstrated consistency with TaqMan qPCR results and adherence to Mendelian inheritance patterns.
• Validated the practicability of cliCRISPR for multiplexed genotyping in family samples.

Conclusions:

• cliCRISPR offers a novel crRNA-limited approach for multiplexed nucleic acid detection.
• The method enables precise genotyping by correlating fluorescence with crRNA concentration.
• cliCRISPR shows significant potential for developing advanced multiplexed biosensors based on CRISPR/Cas12a technology.