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,t ransform, translate,a nd amplify biological signals into physicochemical signals based on logic design principles can be apowerful means for the development of av ariety of biotechnologies.T oe xplore the conceptual validity,w ed esign aC RISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade,w hich probes as pecific biomolecular input, transform the input into as tructurally accessible form for circuit wiring,t ranslate the input information into an arbitrary sequence,a nd finally amplify the prescribed sequence through autonomous formation of as ignaling concatemer.T his upstream biochemical circuit is further wired with ad ownstream electrochemical interface,d elivering an integrated bioanalytical platform. We program this platform to directly analyzethe genome of SARS-CoV-2 in human cell lysate,d emonstrating the capability and the utility of this unique integrated system.

Unified-amplifier based primer exchange reaction (UniAmPER) enabled detection of SARS-CoV-2 from clinical samples

“…Concatemer DNA sequences that were synthesized by PER have been shown to be applicable in a variety of fields 14 , 15 , 17 , 18 , 19 , 20 . Example include detection of a single stranded nucleic acids [16] . The highest yield in PER would guarantee the lowest limit of detection for such analyses.…”

“…SABER in combination with present technologies such as fluorescence in situ hybridization (FISH) made scaffold DNAs that bind fluorescently labeled “imagers” and produced highly elaborate cell images in multiplex formats [4] . In addition, concatemer DNA sequences that have been synthesized by PER have been recently used for detection of macromolecules 14 , 15 , 16 , 17 , 18 , 19 and as a therapeutic agent [20] . In a very recent paper, the genomic RNA of SARS-CoV-2 was detected by combination of CRISPR-Cas9 technology with PER [16] .…”

“…In addition, concatemer DNA sequences that have been synthesized by PER have been recently used for detection of macromolecules 14 , 15 , 16 , 17 , 18 , 19 and as a therapeutic agent [20] . In a very recent paper, the genomic RNA of SARS-CoV-2 was detected by combination of CRISPR-Cas9 technology with PER [16] .…”

“…In the special case of application of PER for detection of nucleic acid molecules, the molecular setup of the reactions consisted of three DNA elements; a primer strand, the detector hairpin and the amplifier hairpin [13] . The detector was also named as translator [16] . In such cases, the detection cycle was distinct from amplification cycles.…”

CRISPR-Cas based virus detection: Recent advances and perspectives

“…CRISPR-Cas systems, as excellent gene editing tools, are also developed as novel and powerful diagnostic techniques (Abudayyeh and Gootenberg, 2021). For example, CRISPR-Cas9 has been utilized to detect viruses through linking Cas9 as a recognition element and expanded its capability by other designs ( Azhar et al, 2021 ; Dai et al, 2020b ; Jiao et al, 2021 ; Lin et al, 2021 ; Pardee et al, 2016 ; Wang et al, 2020d ). Different from CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13 are developed as powered diagnostic tools with their trans -cleavage activity, which have ushered a new era in molecular diagnostics.…”