Rapid and sensitive Cas12a-based one-step nucleic acid detection with ssDNA-modified crRNA

Area of Science:

• Molecular Biology
• Biotechnology
• Diagnostics

Background:

• CRISPR-Cas12a systems offer rapid nucleic acid detection but face challenges like operational complexity, contamination risk, and CRISPR RNA (crRNA) degradation.
• Existing methods require multiple steps and are susceptible to issues that can compromise diagnostic accuracy and efficiency.

Purpose of the Study:

• To develop a simplified, highly sensitive, and stable one-step CRISPR-Cas12a-based diagnostic assay.
• To overcome the limitations of traditional CRISPR RNA (crRNA) by introducing a single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA).

Main Methods:

• Development of a one-step assay (CasDOS) utilizing a modified crRNA (mD-crRNA) with ssDNA extensions.
• Evaluation of mD-crRNA stability against RNase R treatment.
• Testing CasDOS for the detection of Streptococcus agalactiae (GBS), human papillomavirus (HPV) types 16 and 18, and in clinical samples.

Main Results:

• CasDOS demonstrated a 100-1000 fold increase in sensitivity compared to wild-type crRNA, with a detection limit as low as 16.6 aM for target plasmids.
• The assay achieved 100% accuracy in HPV genotyping and showed complete concordance with qPCR for GBS detection in clinical samples.
• CasDOS provided results within 30 minutes and the mD-crRNA showed enhanced stability, indicating suitability for diagnostic kits.

Conclusions:

• CasDOS is a universal, rapid, and highly sensitive one-step CRISPR detection assay.
• The use of mD-crRNA significantly enhances the performance and stability of CRISPR-based diagnostics.
• This method offers a promising alternative for point-of-care nucleic acid testing.