Modular CRISPR/Cas12a synergistic activation platform for detection and logic operations

Abstract

The revolutionary technology of CRISPR/Cas has reshaped the landscape of molecular biology and molecular engineering. This tool is of interest to researchers in multiple fields, including molecular diagnostics, molecular biochemistry circuits, and information storage. As CRISPR/Cas spreads to more niche areas, new application scenarios and requirements emerge. Developing programmability and compatibility of CRISPR/Cas becomes a critical issue in the new phase. Here, we report a redundancy-based modular CRISPR/Cas12a synergistic activation platform (MCSAP). The position, length, and concentration of the redundancy in the split DNA activators can finely regulate the activity of Cas12a. With the redundant structure as an interface, MCSAP serves as a modular plug-in to seamlessly integrate with the upstream molecular network. MCSAP successfully performs three different tasks: nucleic acid detection, enzyme detection, and logic operation. MCSAP can work as an effector for different molecular networks because of its compatibility and programmability. Our platform provides powerful yet easy-to-use tools and strategies for the fields of DNA nanotechnology, molecular engineering, and molecular biology.

Graphical Abstract

Figure 1.

(A) Scheme and (B) fluorescence plot of the synergistic activation pattern of CRISPR/Cas12a based on redundant structures. (C) Scheme and (D) fluorescence plot of the effect of redundancy on complete activators. (E) Scheme and (F) bubble plot of the positional combination pattern of split activators. The fluorescence rate was calculated based on the slope of the fluorescence curves. (G) Comparison of a-1 and a-2 with different redundancy positions. (H) Comparison of inner and outer redundancy positions with different activators. (I) Scheme and (J) fluorescence plot of the effect of crRNA and activator complementary lengths. (K) Scheme and (L) fluorescence plot of the trisection split activators. crRNA-1 and crRNA-2 have different sequences in the spacer domain. (M) Relative rate of fluorescence increase of the competition between activators without (a–n) and with redundancy (a–r). (N) Non-concentration-dominated rate distribution due to affinity differences. (O) scheme of the competition between activators without (a–n) and with redundancy (a–r).

Figure 2.

(A) Schematic diagram of redundancy at different lengths and positions. a2-i-x denotes the inner side of activator-2 with a x nt of random sequences. (B–E) Fluorescence rates of redundancy of different lengths at four different positions. (F–G) Pairwise comparison of the inner side redundancy of activator-1 and activator-2. (H, I) Pairwise comparison of the outer side redundancy of activator-1 and activator-2. (J, K) Pairwise comparisons of inner and outer redundancy. Two-tailed paired t-tests were used for statistical difference analysis. *P value ≤ 0.05; **P value ≤ 0.01; ***P value ≤ 0.001; ****P value ≤ 0.0001.

Figure 3.

(A) Synergistic activation mode for differential concentration. (B) A fixed a1 concentration with different a2 concentrations. (C) A fixed a1 concentration with different a2 concentrations. (D) The same concentration for a1 and a2. (E–G) Comparison of fluorescent rate for different concetration activation modes.

Figure 4.

(A) Schematic diagram of single nucleotide variant detection. (B) Fluorescence plot of Endo IV operating in different buffers. DF (Discrimination factor) was defined as the ratio of fluorescence rate between a1-i/MT and a1-i/WT. (C–E) Optimization of substrate structure of Endo IV. ±x indicates that the target strand is complementary to the probe to the xth base of the 3′-end or 5′-end of the AP site. (F) Fluorescence plot of MCSAP in response to different concentrations of MT. (G) Fluorescence plot of MCSAP in response to different abundances of MT. (H) Diagram and (I) molecular process for the detection of two targets of the CYP1A2 gene. (J) Fluorescence plot of different SNP combinations. (K) Gene polymorphism decoder. (L) Fluorescence plot of weighting coefficients based on redundancy. The weight sum indicates the signal difference between the mismatch and match groups at 25 minutes after the start of the reaction. (M) Weighting coefficients for different redundancy positions and lengths. (N) Decoding of CYP2C19 gene and drug metabolism.

Figure 5.

(A) Schematic diagram of APE 1 detection. (B) Schematic diagram and (C) fluorescence plot of APE 1 preferred substrate structure optimization. (D) Fluorescence plot of MCSAP for detection of different concentrations of APE 1. (E) Selectivity of MCSAP for APE 1 compared with other nucleases. (F) The ability of MCSAP to detect APE 1 in actual biological samples. Extracts of MDA-MB-231 cells that diluted to about one cell was tested. NCA is an APE1-specific inhibitor.

Figure 6.

(A, B) Schematic and molecular implementation of the ‘AND’ gate operation strategy of MCSAP. (C) Optimization of cofactor concentration for 8–17 DNAzyme. (D) 8–17 and (E) 10–23 performing ‘AND’ gate operations. (F) Three-layer structure: Input-Encoder-Effector for processing arbitrary nucleic acid inputs. (G) Schematic diagram, (H) truth table, (I) molecular implementation, and (J) fluorescence plot of ‘AND’ gate. (K) Schematic diagram, (L) truth table, (M) molecular realization, and (N) fluorescence plot of the ‘OR’ gate. (O) Schematic diagram, (P) molecular realization, and (Q) fluorescence plot of a two-level cascade logic gate. The two ‘OR’ gates are the first level, and the ‘AND’ gate is the second level. (R) Schematic diagram, (S) truth table, (T) molecular realization, and (U) fluorescence plot of the ‘NAND’ gate. (V) Schematic diagram, (W) truth table, (X) molecular realization and (Y) fluorescence plot of the ‘XOR’ gate.