Rapid, easy, sensitive, low‐cost and on‐site detection of environmental DNA and RNA using CRISPR‐Cas13

Abstract

Abstract Environmental DNA (eDNA) monitoring of species distribution has become a critical tool in ecology, conservation biology and fisheries for identifying the presence and distribution of diverse organisms, including important, threatened and invasive species. However, eDNA detection still has room for improvement in sensitivity, requiring time‐consuming real‐time polymerase chain reaction (qPCR) steps and costly machines. In this study, we report a CRISPR‐Cas13‐based method for rapid, easy, sensitive, low‐cost and on‐site detection of eDNA. The assay for the detection of common carp (Cyprinus carpio) and medaka (Oryzias latipes) nucleic acid employs a two‐step process, starting with recombinase polymerase amplification (RPA) and followed by cleavage using Cas13 nuclease, to effectively identify mitochondrial DNA or RNA. Our results showed that the Cas13‐based method has a higher sensitivity than the qPCR‐based method in detecting tiny amounts of eDNA. When combined with reverse transcription of environmental RNA (eRNA), our method increased detection sensitivity by approximately one order of magnitude. Cas13‐based detection could achieve on‐site detection of eDNA using a quick nucleic acid extraction solution and lateral flow strips. Cas13‐based detection of eDNA and eRNA requires minimal training efforts and can be performed in 1 h, without the need for centrifugation and qPCR machines. The portability of the Cas13‐based method, along with its accuracy and relative ease in designing primers and CRISPR RNA (crRNA), underscores its potential to broaden the application of eDNA and eRNA in various fields, including biodiversity conservation.

Keywords

• CRISPR‐Cas
• real‐time PCR
• recombinase polymerase amplification
• reverse transcription
• species‐specific detection