ABSTRACT
Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove, and employ this method in conjunction with experimental screening to generate binders for 5 distinct DNA targets. These binders exhibit specificity closely matching the computational models for the target DNA sequences at as many as 6 base positions and affinities as low as 30-100 nM. The crystal structure of a designed DBP-target site complex is in close agreement with the design model, highlighting the accuracy of the design method. The designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method is a substantial step towards a general route to small and hence readily deliverable sequence-specific DBPs for gene regulation and editing.
ABSTRACT
Sexual dimorphism is a device that supports genetic diversity while providing selective pressure against speciation. This phenomenon is at the core of sexually reproducing organisms. Caenorhabditis elegans provides a unique experimental system where males exist in a primarily hermaphroditic species. Early works of John Sulston, Robert Horvitz, and John White provided a complete map of the hermaphrodite nervous system, and recently the male nervous system was added. This addition completely realized the vision of C. elegans pioneer Sydney Brenner: a model organism with an entirely mapped nervous system. With this 'connectome' of information available, great strides have been made toward understanding concepts such as how a sex-shared nervous system (in hermaphrodites and males) can give rise to sex-specific functions, how neural plasticity plays a role in developing a dimorphic nervous system, and how a shared nervous system receives and processes external cues in a sexually-dimorphic manner to generate sex-specific behaviors. In C. elegans, the intricacies of male-mating behavior have been crucial for studying the function and circuitry of the male-specific nervous system and used as a model for studying human autosomal dominant polycystic kidney disease (ADPKD). With the emergence of CRISPR, a seemingly limitless tool for generating genomic mutations with pinpoint precision, the C. elegans model system will continue to be a useful instrument for pioneering research in the fields of behavior, reproductive biology, and neurogenetics.
