Programmable Multiplexed Nucleic Acid Detection by Harnessing Specificity Defect of CRISPR-Cas12a.

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• CRISPR-Cas12a offers advanced nucleic acid detection but faces limitations in distinguishing targets with sequence mismatches.
• Understanding mismatch profiles is crucial for enhancing CRISPR-Cas12a specificity and developing novel diagnostic tools.

Purpose of the Study:

• To investigate CRISPR-Cas12a's tolerance to various mismatch types, quantities, and locations in nucleic acid detection.
• To develop a programmable, multiplexed nucleic acid assay leveraging CRISPR-Cas12a's specificity for sensitive pathogen detection.

Main Methods:

• Investigated CRISPR-Cas12a mismatch profiles to understand its specificity limitations.
• Designed a dual-mode detection strategy for approximate and precise target sequence querying.
• Developed a programmable multiplexed nucleic acid assay and integrated it into a paper-based microfluidic chip.

Main Results:

• The assay achieved attomolar sensitivity for simultaneous detection of 14 high-risk human papillomavirus (HPV) subtypes, responsible for 99% of cervical cancer cases.
• Successfully distinguished between HPV16, HPV18, and 12 other high-risk pooled HPV subtypes.
• Clinical validation using 75 swab samples demonstrated performance comparable to PCR, highlighting the chip's potential for point-of-care testing.

Conclusions:

• The developed programmable multiplexed nucleic acid assay effectively addresses CRISPR-Cas12a specificity limitations for enhanced detection.
• The paper-based microfluidic chip enables sensitive, specific, and simultaneous detection of multiple HPV subtypes, suitable for low-cost point-of-care diagnostics.
• This technology holds significant potential for broad application in the sensitive and specific detection of various pathogens.