An Integrated Multi-Function Heterogeneous Biochemical Circuit for High-Resolution Electrochemistry-Based Genetic Analysis

Abstract

Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify, transform, translate, and amplify biological signals into physicochemical signals based on logic design principles can be a powerful means for the development of a variety of biotechnologies. To explore the conceptual validity, we design a CRISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade, which probes a specific biomolecular input, transform the input into a structurally accessible form for circuit wiring, translate the input information into an arbitrary sequence, and finally amplify the prescribed sequence through autonomous formation of a signaling concatemer. This upstream biochemical circuit is further wired with a downstream electrochemical interface, delivering an integrated bioanalytical platform. We program this platform to directly analyze the genome of SARS-CoV-2 in human cell lysate, demonstrating the capability and the utility of this unique integrated system.

Keywords: CRISPR; bioanalytical chemistry; electrochemistry; genetic circuits; primer exchange reaction.

Figure 1.. Modular construction of a multi-function heterogeneous biochemical reaction circuits.

a) A multi-function heterogeneous biochemical circuit constructed by paired CRISPR system and primer exchange reaction, processing genetic information and translating into electric signal. b) Guided by two offset sgRNAs, a pair of CRISPR Cas9 D10A nucleases target opposite sequence on the gene target, transforming the intact dsDNA into a 3’-overhang strand available for cascading circuit. (Blue triangle: cleavage position; Yellow box: PAM region). c) Primer exchange reaction mediated translator and amplifier. A protector gated hairpin 1 serves as a translator, only functioning with the presence of the gene target. An arbitrary sequence (S) is stored in the nascent strand (P1-S) elongated by hairpin 1. Hairpin 2 serves as an amplifier and catalyses the extension of P1-S with repetitive sequence S, forming a concatemer. d) The output of the heterogeneous biochemical circuit is examined by an electrochemical biosensing platform. A capture strand is tethered on the electrode to probe any synthesized concatemer. A signaling probe containing an electrochemical tag, complementary to the repetitive sequence S, binds to the concatemer and generates electrochemical signal.

Figure 2.. Proof of concept evaluation of the electrochemistry transduced biochemical circuit.

a) Time-dependent electrical outputs based on activated circuit (red), negative control (without gene [blue] and without primer [black]). b) A typical square wave voltammetry (SWV) graph showing the electrochemical currents of 1) fully activated circuit (red) with primer (final concentration of 100 nM) and gene (final concentration of 50 nM); 2) non-activated circuit without gene (blue) or without primer (black). c) The Boole-an AND conjunction dependent circuit elements. CRISPR processor is operated as a 3-input AND gate. PER based translator and amplifier elements are operated as a 4-input AND gate. d) Comparison of the performance of the integrated circuit through electrochemical signal gain %= (peak current-baseline current [without primer condition])/baseline current). Each input element was investigated based on the Boolean logic. *P<0.01; **P<0.05 (signal of fully operated circuit against signal of incomplete circuit). The bar represents the mean value of three orthogonal repeats. The error bar represents ± SE.

Figure 3.. Two-pair CRISPR processed target gene multiplexed signaling pathway.

a) Four separate sgRNAs direct four Cas9 D10A nucleases to adjacent target sites, transforming the target into three fragments containing two distinct 3’overhangs accessible for molecular cascading. b) Two orthogonal translator hairpins are initiated by available target sites resulted from CRISPR processing. Target information is translated into an arbitrary sequence, which is further integrated into the same amplifier hairpin, producing a signaling concatemer. c) Comparison of signal gain between single signaling pathway and multiplexed signaling pathway based on different concentrations of gene input. Signal gain %=(peak current-baseline current[without gene condition])/baseline current). d) A dose-dependent electrochemical response of integrated biochemical circuit in a range of concentrations of gene input. The bar represents the mean value of three orthogonal repeats. The error bar represents ± SE. **P<0.05.

Figure 4.. A modular bioanalytical strategy.

a) The modularity nature of the biochemical circuit allows simple construction of individual processes for sample analysis, transforming biomolecule input into electrical output within 2 hr. b) Evaluation of the matrix effect of the bioanalytical platform with spiked samples in human cell lysate (green) proved the capability of the platform on complex sample analysis. Interference evaluation based on non-specific gene target (HPV-16) demonstrated the reliable selectivity of the CRISPR mediated recognition process.