Abstract
With the trend of moving molecular tests from clinical laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established diagnostics with the ease, cost effectiveness and speed of isothermal amplification and detection methods. A promising new nucleic acid detection method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.
Highlights
The access to rapid and reliable detection methods of nucleic acids is critical in many different fields, such as life sciences, environmental monitoring, biotechnology and maybe most importantly health care
Class 2 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems are characterized by a single multido main protein that associates with an RNA sequence to form a ribonu cleoprotein (RNP) surveillance complex
The Cas13 effector proteins do not require a protospacer adjacent motif (PAM) sequence, but have a protospacer flanking site (PFS) where a guanine base directly following the protospacer has a negative effect on complementation between CRISPR RNA (crRNA) and target single stranded RNA (ssRNA), while any other nucleotide does not influence the enzymatic activity (Omar O Abudayyeh et al, 2016)
Summary
The access to rapid and reliable detection methods of nucleic acids is critical in many different fields, such as life sciences, environmental monitoring, biotechnology and maybe most importantly health care. Sensing of pathogens on the basis of their genetic information, for instance by monitoring circulating cell-free DNA/RNA particles related to various types of diseases makes early diagnosis and treatment possible This depends on in methods that allow the detection of ultralow concentrations of nucleic acids with high sensitivity and specificity. There is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established genomic diagnostics, with the ease, cost effectiveness and speed of isothermal amplification methods Such a newly developed diagnostic tool will be perfect for point-of-care (POC) testing. Class 2 CRISPR/Cas systems are characterized by a single multido main protein that associates with an RNA sequence to form a ribonu cleoprotein (RNP) surveillance complex This RNA sequence is called guide RNA (gRNA). These structural changes ‘activate’ the effector protein and induce the formation of the RNP surveillance complex, which scans nucleic acids and targets sequences complemen tary to its crRNA for enzymatic degradation
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