Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

Long T. Nguyen; Brianna M Smith; Piyush K. Jain

Using ENHANCE for detecting the PCA3 cDNA, the limit of detection was determined to be as low as 25 fM in the urine at 13 mM Mg 2+ concentration compared to ~1 pM at 3 mM Mg 2+ concentration after 6 hours (Figs. 3a-c and Supplementary Figs. 17-19 ). In contrast, the wild-type crRNA also showed a similar 29 fM limit of detection at 13 mM Mg 2+ concentration while the limit of detection was ~10 pM at 3 mM Mg 2+ concentrations after 6 hours. Therefore, by combining the crRNA modifications with increased Mg 2+ ion concentrations, we achieved approximately 400-fold increase in sensitivity, based on limit of detection calculations. Nevertheless, this observation also suggests that our modified crRNA+3'DNA7 significantly improves the limit of detection at low Mg 2+ but reaches a saturation point that is comparable with the wild-type crRNA at high Mg 2+ concentration. To understand the importance of divalent ions in the Cas12a transcleavage reaction, we carried out a Michaelis-Menten kinetic study with various Mg 2+ concentrations ( Supplementary Fig. 17 ). We observed that the initial reaction rate of Cas12a in the presence of high Mg 2+ concentrations increased tremendously compared to that in low Mg 2+ . ssDNA. Using modified crRNA, the limit of detection of HCV target ssDNA was found to be 290 fM (29 amoles) at 30 min, without target amplification, mean ± SE, two-way ANOVA (n=3, N=3). (i) Fold change in trans-cleavage activity with LbCas12a in presence of 100 pM (10 fmols) of target SARS-CoV-2 cDNA (dsDNA), mean ± SE, two-way ANOVA (n=3, N=2, ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (j) Lateral flow assay detecting 1 nM (100 fmols) of SARS-CoV-2 cDNA using either crCoV-2 or crCoV-2+3'DNA-7 within 20 minutes of incubation. (j) Schematic diagram showing how a lateral flow assay works. Briefly, the dipstick uses gold-labeled FITC-specific antibodies that binds to FITC-biotin reporter and travel through membrane. Only cleaved reporter will reside at the positive line. (k) Lateral flow assay detecting SARS-CoV-2 cDNA using crCov-2 and crCoV-2+3'DNA7 without a pre-amplification step, and (l) band-intensity analysis of (k) using ImageJ. (m) Lateral flow assay detecting SARS-CoV-2 RNA N gene using crCov-2 and crCoV-2+3'DNA7 with RT-LAMP, and (n) band-intensity analysis of (m) using ImageJ. author/funder. All rights reserved. No reuse allowed without permission.