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3.4.2.3 Biochemical Detection
ОглавлениеFinally, we would like to point that CRISPR systems can be used for molecular diagnostics. The collateral activity of Cas13 proteins, while conceptually undesirable, has been exploited to develop highly sensitive detectors for the presence of specific RNA. This approach relies on in vitro activation of Cas13’s indiscriminate RNase activity by an on‐target recognition of specific RNA. Once activated, Cas13 is able to degrade other RNA molecules in the reaction mix; if a fluorescent RNA reporter molecule is included, then Cas13 is able to detect a specific RNA molecule with attomolar sensitivity (Gootenberg et al. 2017), and improvements claiming to bring sensitivity down to zeptomolar (10−21 mol/l) range (Gootenberg et al. 2018) (Figure 3.7h). This system, termed SHERLOCK, has been used since to develop fast and sensitive methods for diagnostics of a number of highly pathogenic RNA viruses (Myhrvold et al. 2018; Patchsung et al. 2020). A similar approach exploits a collateral activity of Cas12a proteins on ssDNA, allowing one to detect specific DNA (or cDNA) sequences; indeed a flurry of different variants of this concept have been developed to detect pathogen DNA or cDNA or to perform SNP profiling (Chen et al. 2018; Li et al. 2018b; Teng et al. 2019b). These developments and innovation are momentous and provide a major progress for molecular diagnostics (Li et al. 2019b).