CRISPR-Assisted Genetic Engineering

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are a genomic feature of many prokaryotic and archaeal species. CRISPR functions as an adaptive immune system, targeting exogenous DNA or RNA sequences that match 30-40bp spacers integrated into the genome. Our project focuses on developing a set of tools for synthetic control over the CRISPR pathway. This includes a method for creating polymers of repeat-spacer-repeat arrays, the development of CRISPR Biobricks (CAS genes, leader sequences, and RSR arrays) for several sub-types (E. coli, B. halodurans, and L. innocua), tests of these components on plasmids containing GFP, and a software tool to collect and display CRISPR information as well as select spacers from a user-determined sequence. Both DNA-targeting and RNA-targeting systems were explored. Preliminary tests of a synthesized RSR array in E. coli K-12 MG1655 show a 30-fold increase in transformation efficiency. Given the relatively recent progress in the scientific understanding of this system, we see the potential for a wide range of biotechnological applications of CRISPR in the future. These include: in vitro characterization of the CRISPR-Cas system, combinatorial gene silencing using custom RNA-targeting constructs, and directed evolution using CRISPR as a driving force for mutating a gene of interest.