Transplanting a Bacterial Immune System: Determining the Function of a Novel CRISPR System

CRISPR (Clusters of Regularly Interspaced Short Palindromic Repeats) loci and cas (CRISPR-associated) genes provide adaptive immunity (see panel below) in bacteria and have recently been repurposed for genome editing. Systems are structurally and functionally diverse. 2 classes, 6 types, 33 subtypes Very few have been studied experimentally None of the Type IV systems have been characterize

Transplanting a Bacterial Immune System: Design, Construction, and Expression of a Multi-subunit Type IV CRISPR System

CRISPR (Clusters of Regularly Interspaced Short Palindromic Repeats) loci and cas (CRISPR associated) genes provide adaptive immunity against invasive elements such as viruses and plasmids in bacteria and archaea. These RNA-guided systems have been repurposed for wide applications in genome engineering and have the potential to cure genetic diseases. Although the structure and function of CRISPR system Types I, II, III, V, and VI have been determined, the Type IV CRISPR systems have not yet been examined experimentally. To better understand the structure and function of Type IV CRISPR systems, we have constructed a plasmid to recombinantly co-express several Type IV CRISPR system proteins from Acidothiobacillus ferrooxidans. We hypothesize that recombinant co-expression in E. coli of all the Type IV genes (csf1, csf2, csf3, and cas6) along with a CRISPR will allow for a multi-subunit RNA-guided surveillance complex to form in vivo. Using ligation-independent cloning, Type IV genes csf1, csf2, csf3, and cas6 were inserted into transfer vectors with no tag, a Histidine-tag, and a Maltose Binding Protein tag. Traditional cloning methods were used to move sequences from the transfer vectors into destination vectors to construct multiple iterations of a polycistronic vector. With these constructs in hand, tests to determine optimal growth conditions for expression of the multi-protein Type IV CRISPR system complex are ongoing