Active CRISPR-Cas12a on Hydrophilic Metal–Organic Frameworks: A Nanobiocomposite with High Stability and Activity for Nucleic Acid Detection

Abstract

CRISPR-Cas12a is an accurate and responsive biosensing technique, but its limited stability has restricted its widespread applications. To address this, we propose a strategy using metal−organic frameworks (MOFs) to protect Cas12a from harsh environments. After screening multiple candidate MOFs, it was found that hydrophilic MAF-7 is highly compatible with Cas12a, and the as-formed Cas12a-on-MAF-7 (COM) not only retains high enzymatic activity but also possesses excellent tolerance to heat, salt, and organic solvents. Further investigation showed that COM can serve as an analytical component for nucleic acid detection, resulting in an ultrasensitive assay for SARS-CoV-2 RNA detection with a detection limit of 1 copy. This is the first successful attempt to create an active Cas12a nanobiocomposite that functions as a biosensor without the need for shell deconstruction or enzyme release.

PCR Independent Strategy-Based Biosensors for RNA Detection

“…Instead of using preamplification to achieve signal amplification, Zhi Run Ji et al [111] proposed a strategy to use metal-organic frameworks (MOFs) to protect Cas12a from harsh environments and successfully constructed an active Cas12a nanocomposite, Cas12a-on-MAF-7 (COM), as a biosensor for the first time, without the need of isothermal amplification. This strategy achieved an ultra-sensitive SARS-CoV-2 RNA detection with a detection limit of one copy and solved the problem of poor stability of CRISPR-Cas12a (Figure 6D).…”

“…(C) Schematic of SARS-CoV-2 DETECTR workflow[106]. (D) Cas12a assembled with MAF-7 to form nanobiocomposites with higher stability and trans-cleavage activity for nucleic acid testing[111]. (E) Schematic illustration of SATORI.…”

Encapsulation of Enzymes into Hydrophilic and Biocompatible Metal Azolate Framework: Improved Functions of Biocatalyst in Cascade Reactions and its Sensing Applications

“…MAF-7 consisting of zinc ion (metal source) and 3-Methyl-1H-1,2,4-triazole (Hmtz) (organic ligand) endows sufficiently high hydrophily. [26,38] It can be transferred efficiently in aqueous phase, demonstrating remarkable biocompatibility to load different proteins. Accordingly, the curiosity that whether MAF-7 could be employed as another excellent carrier to encapsulate enzymes is evoked due to the unique functions it emerged.…”

DNA Robots for CRISPR/Cas12a Activity Management and Universal Platforms for Biosensing

“…The trans -cleavage of Cas12a is multiple turnover, thereby enabling the continuous cleavage of the ssDNA substrate to achieve signal amplification . These unique activities make the Cas12a system a good signal transduction element that can perform both biometric events and signal conversion output, and amplify the output signal to facilitate sensitive detection . Based on this, precisely and controllably managing the cleavage behavior of the CRISPR/Cas12a system has become a key component in developing excellent biosensing methods.…”