Probable hindrance of visible colour due to excess biotin with CRISPR-dCas9-sgRNA lateral flow assay detection of HPV16 and HPV18: a negative finding.

To develop a CRISPR-dCas9-based lateral flow assay (LFA) for the detection of human papillomavirus 16 (HPV16) and human papillomavirus 18 (HPV18) genotypes for point-of-care molecular diagnosis. A CRISPR-dCas9-based LFA was planned to be developed by immobilizing biotin-tagged dCas9-sgRNA assembly on streptavidin-coated nitrocellulose membranes. The assay protocol involved the sequential addition of biotinylated HPV16 and HPV18 PCR amplicons, streptavidin-alkaline phosphatase conjugate, and BCIP/NBT substrate for colorimetric detection. No colour development was observed in the experimental setup, in contrast to the positive control (biotin, streptavidin-alkaline phosphatase conjugate and BCIP/NBT substrate). The failure was attributed to steric hindrance caused by excess biotin present on both the dCas9-sgRNA complex and the HPV PCR amplicons, leading to competition for streptavidin binding sites, improper binding configurations, disrupted protein folding, and interference with biotin-streptavidin interactions. The study demonstrates that using multiple biotinylated molecules in CRISPR-dCas9-based LFAs can lead to assay failure due to competitive binding and steric hindrance. The results advocate the use of two different molecule for immobilization and signal generation and emphasize the necessity for independent optimization of binding and reporting components to establish a functional CRISPR-dCas9-based LFA platform.