Development and application of sensitive, specific, and rapid CRISPR‐Cas13‐based diagnosis

Shihong Gao, David; Zhu, Xiaodong; Lu, Binfeng

Nucleic acid detection is a necessary part of medical treatment and fieldwork. However, the current detection technologies are far from ideal. A lack of timely and accessible testing for identifying cases and close contacts has allowed severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2), the causative virus of the ongoing coronavirus disease‐2019 (COVID‐19) pandemic, to spread uncontrollably. The slow and expensive detection of mutations—predictors for chronic diseases such as cancer—form a barrier to personalized treatment. A recently developed diagnostic assay is ideal and field‐ready—it relies on CRISPR‐Cas13. CRISPR‐Cas13 works similarly to other CRISPR systems: Cas13 is guided by a crRNA to cleave next to a specific RNA target sequence. Additionally, Cas13 boasts a unique collateral cleavage activity; collateral cleavage of a fluorescent reporter detects the presence of the target sequence in sample RNA. This system forms the basis of CRISPR‐Cas13 diagnostic assays. CRISPR‐Cas13 assays have >95% sensitivity and >99% specificity. Detection is rapid (<2 h), inexpensive ($0.05 per test), and portable—a test using lateral flow strips is akin to a pregnancy test. The recent adaptation of micro‐well chips facilitates high‐level multiplexing and is high‐throughput. In this review, we cover the development of CRISPR‐Cas13 assays for medical diagnosis, discuss the advantages of CRISPR‐Cas13‐based diagnosis over the traditional reverse transcription polymerase chain reaction (RT‐PCR), and present examples of detection from real patient samples.