A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics

Abstract

Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas–powered catalytic nucleic acid circuit, namely, CRISPR-Cas–only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer–associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.

Advances in the Integration of Nucleic Acid Nanotechnology into CRISPR-Cas System

“…And our Reproduced with permission from Ref. [71]. Copyright 2020, The American Association for the Advancement of Science work suggested that the integration of nucleic acid circuit and CRISPR-Cas systems could be an effective strategy to develop sensitive CRISPR-based bioassays [70].…”

“…8c). Recently, we presented a CRISPR-Cas-powered catalytic nucleic acid circuit for isothermally amplified detection of genomic DNA [71]. By rationally designing a switchable sgRNA with signal reporting capacity, we constructed a Cas12a-based positive feedback circuit with exponential dynamic, which achieved the effective detection of hepatitis B virus infection and human bladder cancer-associated single-nucleotide mutation in clinical samples (Fig.…”

CRISPR-Cas based virus detection: Recent advances and perspectives

“…Using the platform, multiple clinically relevant nucleic acid targets were detected and the limit of detection reached femtomolar without any pre-amplification ( Nguyen et al, 2020 ). Besides, Shi et al designed a CRISPR-Cas12a powered positive feedback circuit and the HBV DNA was detected with attomolar sensitivity ( Shi et al 2021 ).…”

Specific Versus Non‐specific Response in Exponential Molecular Amplification from Cross‐Catalysis: Modeling the Influence of Background Amplifications on the Analytical Performances

“…A prototypical example is the well-known Polymerase Chain Reaction (PCR), [15] but isothermal alternatives are now widely developed, among which Exponential Amplification Reaction (EXPAR) is particularly efficient. [16][17][18][19][20] For non-nucleic acid targets, it is possible to combine the high amplification power of PCR or EXPAR with the specificity of immunoassays, such as in immuno-PCR [21,22] or immuno-EXPAR. [23,24] However, alternatives to nucleic acid-based exponential amplification are very rare and it is a problem for target lacking immunogenicity such as small molecules.…”

“…It is particularly true for the detection of nucleic acids, for which numerous strategies based on template replication have been developed. A prototypical example is the well‐known Polymerase Chain Reaction (PCR), [15] but isothermal alternatives are now widely developed, among which Exponential Amplification Reaction (EXPAR) is particularly efficient [16–20] . For non‐nucleic acid targets, it is possible to combine the high amplification power of PCR or EXPAR with the specificity of immunoassays, such as in immuno‐PCR [21,22] or immuno‐EXPAR [23,24] .…”