Strategies to Improve Multi-enzyme Compatibility and Coordination in One-Pot SHERLOCK

Abstract

While molecular diagnostics generally require heating elements that supply high temperatures such as 95 °C in polymerase chain reaction and 60–69 °C in loop-mediated isothermal amplification, the recently developed CRISPR-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can operate at 37 °C or a similar ambient temperature. This unique advantage may be translated into highly energy-efficient or equipment-free molecular diagnostic systems with unrestricted deployability. SHERLOCK is characterized by ultra-high sensitivity when performed in a traditional two-step format. For RNA sensing, the first step combines reverse transcription with recombinase polymerase amplification, while the second step consists of T7 transcription and CRISPR-Cas13a detection. The sensitivity drops dramatically, however, when all these components are combined into a single reaction mixture, and it largely remains an unmet need in the field to establish a high-performance one-pot SHERLOCK assay. An underlying challenge, conceivably, is the extremely complex nature of a one-pot formulation, crowding a large number of reaction types using at least eight enzymes/proteins. Although previous work has made substantial improvements by serving individual enzymes/reactions with accommodating conditions, we reason that the interactions among different enzymatic reactions could be another layer of complicating factors. In this study, we seek optimization strategies by which inter-enzymatic interference may be eliminated or reduced and cooperation created or enhanced. Several such strategies are identified for SARS-CoV-2 detection, each leading to a significantly improved reaction profile with faster and stronger signal amplification. Designed based on common molecular biology principles, these strategies are expected to be customizable and generalizable with various buffer conditions or pathogen types, thus holding broad applicability for integration into future development of one-pot diagnostics in the form of a highly coordinated multi-enzyme reaction system.

Rapid detection of mexX in Pseudomonas aeruginosa based on CRISPR-Cas13a coupled with recombinase polymerase amplification

“…However, in this study, the RPA-Cas13a-LFD detection method of P. aeruginosa DNA is only appropriate for the two-step RPA-Cas13a ( Figure 9 ). The one-tube SHERLOCK formulation is an extremely complex and packed molecular system with numerous enzymes in its reaction components ( Li H. et al, 2023 ). For the RPA reaction, a polymer known as polyethylene glycol (PEG) is required for the RPA reaction, which creates a crowded and extremely sticky environment for the RPA protease, interfering with the flow of other components in the experiments and being detrimental for other enzyme activities.…”

Nanotechnology’s frontier in combatting infectious and inflammatory diseases: prevention and treatment

“… 722 – 724 This highlights its potential as an important next-generation detection technology. 725 , 726 …”