One-Pot Molecular Diagnosis of Acute Hepatopancreatic Necrosis Disease by Recombinase Polymerase Amplification and CRISPR/Cas12a with Specially Designed crRNA

Abstract

Acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating diseases in aquaculture, causing significant economic losses in seafood supplies worldwide. Early detection is critical for its prevention, which requires reliable and fast-responding diagnosis tools with point-of-care testing (POCT) capacity. Recombinase polymerase amplification (RPA) has been combined with CRISPR/Cas12a for AHPND diagnosis with a two-step procedure, but the operation is inconvenient and has the risk of carryover contamination. Here, we develop an RPA-CRISPR one-pot assay that integrates RPA and CRISPR/Cas12a cleavage into simultaneous reactions. Using the special design of crRNA, which is based on suboptimal protospacer adjacent motifs (PAM), RPA and Cas12a are made compatible in one pot. The assay is highly specific with a good sensitivity of 102 copies/reaction. This study provides a new choice for AHPND diagnosis with a POCT facility and sets a good example for developing RPA-CRISPR one-pot molecular diagnosis assays.

A PAM-Free One-Step Asymmetric RPA and CRISPR/Cas12b Combined Assay (OAR-CRISPR) for Rapid and Ultrasensitive DNA Detection

“…For instance, some assays employed temporary blocking of crRNA using photocontrolled materials, resulting in ultra sensitivity but falling short of being true one-step methods. ,, Other approaches focused on optimizing the concentrations of reaction components to achieve a one-step RPA-CRISPR/Cas assay, − which, although relatively straightforward to operate, necessitated extensive optimization efforts. Moreover, researchers explored the utilization of suboptimal PAM-site crRNAs ,, or strategically positioning crRNAs adjacent to RPA primers to strike a balance between amplification efficiency and fluorescence signaling. − While these assays have demonstrated commendable performance, their reliance on crRNA design posed challenges and hindered the optimization of crRNA selection. In contrast, our OAR-CRISPR assay obviates the need for a PAM recognition site, substantially expanding the repertoire of crRNA options.…”

“…Despite numerous CRISPR/Cas-assisted assays reported in recent years, only a handful of single-step CRISPR/Cas12-assisted RPA assays have been documented to date. Much of this limitation can be attributed to the low reaction compatibility between these two assays, resulting in poor sensitivity. , One potential explanation for this is the excessive activation of Cas12 by crRNA with a canonical PAM motif, overwhelming the RPA amplification and compromising the synergy between these two reactions. Furthermore, the presence of the PAM motif (e.g., TTN or TTTN) in the target dsDNA is essential for the Cas12-based method .…”

Emerging technologies revolutionising disease diagnosis and monitoring in aquatic animal health

“…Wang et al (2023) developed a one‐pot CRISPR‐Cas12a assay, integrating RPA and CRISPR/Cas12a in a single tube for acute hepatopancreatic necrosis disease (AHPND) detection with sensitivity of up to 100 copies per reaction. Cas12a's optimal cleavage temperature is 37°C, compatible with RPA (37–42°C) 140 . Remote conditions can utilise human body temperature for pre‐amplification and cleavage.…”

Ultrasensitive one-pot detection of monkeypox virus with RPA and CRISPR in a sucrose-aided multiphase aqueous system

“…The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas12a system has been widely applied to the early diagnosis of pathogen infections ( 18 – 20 ). The system relies on the recognition of a pathogen-specific sequence by the Cas12a-crRNA complex followed by the activation of Cas12a to cleave a signal-producing probe.…”