Development of a Crispr/Cas12a-Based Method to Detect Invasive Aquatic Species

Abstract

To establish an invasive aquatic species detection method with high sensitivity and specificity, we used the CRISPR/Cas12a system. After comparing the mitochondrial COX1, COX2, COX3, and CYTB sequences of many invasive species, we designed and synthesized guide RNA (gRNA) and amplified target sequences for a detection system based on binding of the Cas12a protein. We then used alligator gar target DNA as a standard to assess the detection system. The reaction system was optimized in terms of Cas12a and gRNA concentrations, ssDNA reporter concentration, and reaction temperature, and this optimized system was used to detect other invasive species. The results showed that 100 nmol/L Cas12a, 100 nmol/L gRNA, and 700 nmol/L ssDNA reporter added to 20 µL of a DNA sample at 42℃ was optimal, with the system able to detect 10.3 fM target DNA of an invasive species with good specificity and sensitivity. The results were directly observed under UV light irradiation. Moreover, we applied eDNA, which can trigger the generation of fluorescent signals in the system, and the denaturation-annealing-extension cycles of PCR were optimized so that the detection system works when the cycle number is as low as 25 and even when the eDNA was diluted 10-24-fold. Our findings indicate that the established method is effective for the detection of invasive aquatic species.

Keywords: CRISPR-Cas12a, Aquatic animals, Biological invasion, Nucleic acid detection