Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor

Abstract

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor β1 (TGF-β1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.

Keywords: CRISPR Cas12a (cpf1); bioanalytical chemistry; biosensor; electrochemistry; trans-acting cleavage.

Figure 1.. Principle of E-CRISPR.

A) Cas12a (cpf1) performs crRNA guided cis-cleavage (specific target) initiated trans-cleavage activity (nonspecific ssDNA). B) Nonspecific ssDNA reporter with methylene blue tag immobilized on the gold electrode. C) With the presence of the target, Cas12a-crRNA would initiate the trans-cleavage activity on nonspecific ssDNA reporter, resulting a low electrochemical current of methylene blue. D) Without the presence of the target, Cas12a-crRNA would not initiate the trans-cleavage activity on nonspecific ssDNA reporter, resulting a high electrochemical current of methylene blue. E) A representation of electrochemical current outputs based on the without & with target conditions.

Figure 2.. Optimization of on-chip trans-cleavage activity.

A) Representation of square wave voltammetry (SWV) evaluation of E-CRISPR in response to HPV-16. Red curve represents the background signal of 50 nM of Cas12a-crRNA duplex. Black curve represents the signal generated by the 50 nM of Cas12a-crRNA-target induced trans-cleavage activity. B) Evaluation of 50 nM of Cas12a orthologs from Lachnospiraceae bacterium and Acidaminococcus sp on its activity for on-chip trans-cleavage activity based on the change of current between background signal and target-mediated signal. $\Delta \mathrm{I}\%=\frac{\text{Background signal}-\text{Target signal}}{\text{Background signal}}$. (Red line- LbCas12a; Black line- AsCas12a) C) Evaluation of trans-cleavage activity using 50 nM of LbCas12a-crRNA-target triplex. D) Evaluation of the effect of the concentration of divalent metal ions on the trans-cleavage activity of RuvC domain based on 50 nM of LbCas12a-crRNA-target triplex. E) & F) & G) SWV graphs of different lengths of surface ssDNA reporters based on 30 nM of LbCas12a-crRNA-target triplex. H) Comparison of signal change from different lengths of surface ssDNA reporters. SWV graphs in these figures present the result of a single test. Error bars in figures present the standard error (SE) based on at least three individual trails using at least three different sensors.

Figure 3.. E-CRISPR analysis of HPV-16.

A) Dose-response curve of the detection of HPV-16 in different matrixes (green line-10 mM Tris buffer containing 50 mM NaCl and 15 mM MgCl₂; purple line-100% human serum). B) Selectivity study through comparison of the signal changes based on non-target nucleic acids (500 nM) with that of 1 nM of HPV-16 (n=3, *P<0.01, target signal vs. non-target signal). C) Target strands with mismatches at different positions, including PAM region and crRNA complement at different positions: 1, 6, 11, 16. D) Evaluation of the influence of mismatches at different positions on the E-CRISPR signal. A target concentration of 1 nM was applied for all the targets (wild type (WT) and mismatched targets).

Figure 4.. E-CRISPR cascade for protein detection.

A) Sample containing protein target of interest is firstly treated by a fixed concentration of target specific aptamer (ssDNA). B) A E-CRISPR system is specifically designed for the recognition of the aptamer. The remaining concentration of aptamer is analyzed by E-CRISPR. C) A representation of SWV results based on the with target and without target condition. D) Linear calibration curve of TGF-β1 protein detection with an equation of Y=0.91X+1.79 and R-square value of 0.99 (n=3, SE=1.54%). E) Selectivity study through comparison of the signal outputs based on non-target proteins (10 nM) with that of 10 nM of TGF-β1 (n=3, **P<0.01 versus different interference substances). F) Concentration-dependent signals observed within conditioned medium harvested at two time-points during the chondrogenic differentiation program of human mesenchymal stem cells (hMSCs) containing TGF-β1. The samples were analyzed by three sets of individual experiments using three different sensors (n=3, ***P<0.05, Day 28 vs. Day 2). The horizontal black dashed line represents the average signal variation (n=3) based on the presence of blank conditioned medium.