CRISPR-Cas enzymes: The toolkit revolutionizing diagnostics.

The programmable nature of sequence-specific targeting by CRISPR-Cas nucleases has revolutionized a wide range of genomic applications and is now emerging as a method for nucleic acid detection. We explore how the diversity of CRISPR systems and their fundamental mechanisms have given rise to a wave of new methods for target recognition and readout. These cross-disciplinary advances found at the intersection of CRISPR biology and engineering have led to the ability to rapidly generate solutions for emerging global challenges like the COVID-19 pandemic. We further discuss the advances and potential for CRISPR-based detection to have an impact across a continuum of diagnostic applications.

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform.

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.