The CRISPR-Cas system, initially discovered/re-invented for gene editing purposes has found use in the field of nucleic acid detection. The Cas/gRNA complex binds, with high sensitivity, to complementary target nucleic acids of interest leading to a change in the conformation of the Cas protein. This change in conformation subsequently results in the activation of a collateral cleavage state, where the Cas/gRNA system indiscriminately shreds ssDNA. The shredding of ssDNA can be leveraged to perform molecular detection with a ssDNA fluorescent reporter. Unfortunately, since the change in function is associated with a change in conformation, as with other proteins, the process is very sensitive to temperature. This is a drawback, since CRISPR-Cas systems are often used in conjunction with amplification techniques (such as LAMP or PCR). Hui Wu and collaborators from ETH, Zhejiang University and the Guangzhou Laboratory have utilized this Cas/gRNA complex for molecular detection purposes in a novel fluidic system to circumvent these thermal drawbacks.
Their approach utilizes pico-injection (voltage-driven injection of small volumes into droplets) technology to perform LAMP and a subsequent CRISPR reaction in droplets on a single chip. Previously, it was not possible to perform LAMP and a subsequent CRISPR reaction in droplets due to the discrepancy in thermal requirements of Cas/gRNA (37℃) and LAMP (65℃). By solving such temperature considerations, Wu and co-workers were able to leverage the various advantages of containing molecular machinery in droplets, including minimization of adsorption of molecular machinery to the channel walls and compartmentalization of assay components. The authors then used compartmentalization to perform a digital assay. Significantly, the combination of droplet compartmentalization, nucleic acid amplification and CRISPR-based reactions allowed the team to detect the invA gene of Salmonella typhimurium with fM detection limits.
The full paper can be found here.