CRISPR-Cas technology has evolved rapidly from a bacterial adaptive immune system to transformative use in molecular diagnostic and genomic engineering. Beyond traditional genome-editing capabilities, newly engineered versions of CRISPR/Cas can be used for programmable transcriptional regulation, epigenetic modification, molecular imaging, and ultrasensitive nucleic acid detection. Specifically, catalytic-inactive Cas proteins like dCas9 and dCas12 retain their ability to bind specific sequences on DNA but do not cleave it. Therefore, these proteins can be reversibly regulated by either CRISPRi or CRISPRa to alter gene expression. Thus, they represent powerful tools for both functional genomic studies and synthetic biological applications. Advances in CRISPR engineering have recently greatly increased the diagnostic potential of Cas12 and Cas13 enzymes. For example, collateral cleavage activity allowed the creation of CRISPR-based diagnostic platforms (SHERLOCK, DETECTR and FELUDA), which can detect target DNA/RNA sequences at high sensitivity and specificity. Moreover, they were demonstrated to work in detecting several infectious pathogens (SARS-CoV-2, Zika virus, and M. tuberculosis) and thus have significant value in point-of-care testing, especially when there is limited availability of resources. CRISPR systems are also being combined with increasing frequency with epigenetic regulators, fluorescence microscopy methods, biosensors, and lab-on-a-chip platforms that incorporate microfluidics to provide improved molecular analysis and automated diagnosis. The purpose of this review is to describe how engineered CRISPR-Cas systems have been developed from primarily genome editing tools into multi-functional platforms for transcriptional regulation, epigenetic engineering, diagnostics, imaging, and emerging microfluidic integrations. Additionally, this review will address some of the current challenges that exist with using CRISPR-based technologies, including off-target effects, delivery efficiency, diagnostic standardization, scaling up production, and translating these technologies clinically.
