Characterization and biotechnological applications of programmable nucleases
CRISPR systems are immune systems utilized by bacteria and Archaea to protect themselves from invading phages and other foreign nucleic acid. These systems typically use a single subunit or multi-subunit protein complex loaded with a nucleic acid guide that contains a target sequence to find and cleave with a nuclease domain. This activity has led to the use of guides targeting genomic DNA sequences to perform genome editing in native and non-native organisms such as higher eukaryotes.
Ryan Fuchs and his team, Jennifer Curcuru and Meg Mabuchi, have been conducting studies to sample and characterize the natural diversity of different Cas systems. Cas nucleases encoded in different organisms have various properties that could make them uniquely useful for a given application. For instance, they have different requirements in flanking sequences required for cleavage of a target (known as a PAM sequence) and different temperature requirements leading to optimized genome editing in organisms that live at those temperatures. Additionally, we evaluate engineered versions of Cas nucleases and emerging new classes of guided nucleases.
Diagram of SpyCas9 ribonucleotide protein complex bound to target DNA
The modular nature of RNA-guided CRISPR-Cas nucleases makes them attractive in pharmaceutical and biotechnological applications, such as base editing, prime editing, gene knocking down and activation, transposition, and so on. Some types of Cas nucleases also cleave nucleic acids they encounter in trans after they bind their guide-determined target sequence. This property can be utilized to detect nucleic acids of interest by supplying a nucleic acid substrate in trans that generates a signal upon cleavage. Methods based on such nucleases, including Cas12a nucleases, have been used to successfully detect various targets, including bacteria, viruses, and eukaryotic cells. Their innate high specificity and sensitivity are especially useful for rapid point-of-care diagnostic applications, which are instrumental in public healthcare and disease prevention and management. Juan Pan has leveraged the unique features of Cas12a variants to develop novel biosensing technologies for nucleic acid detection.
