Project Summary CRISPR-Cas loci consist of short DNA repeats separated by equally short sequences (known as spacers) that match the genomes of prokaryotic viruses (phages) and plasmids and confer sequence-based immunity against these elements. Immunity is mediated by small, antisense CRISPR RNA molecules (crRNAs) that are transcribed from spacers and guide CRISPR-associated (Cas) nucleases to the invading nucleic acid for cleavage and destruction. During my post-doctoral studies I pioneered the study of CRISPR-Cas systems to establish the foundations of this bacterial immunity pathway. Using genetics, I determined that CRISPR-Cas systems target DNA molecules in a sequence-specific manner, a study that was key to understand the mechanisms of CRISPR immunity at the molecular level. This finding predicted the existence of RNA- programmable Cas nucleases and their current applications to genome editing. Upon plasmid or phage infection, CRISPR-Cas system incorporate new spacer sequences that match the genome of the invader. This process records a memory of the infection that is subsequently used to generate the crRNA guides for the Cas nucleases. While the molecular mechanisms behind the recognition and cleavage of target sequences by the Cas nucleases are well understood, how the host can acquire new spacers; i.e. the immunization phase of the CRISPR-Cas immune response, is still a mystery. In this proposal I plan to study how the prokaryotic host acquires new spacer sequences from its invaders, using a combination of molecular genetics and next-generation sequencing approaches. Fundamental questions such as (i) how autoimmunity, or the acquisition of spacers from the host chromosome, is prevented; (ii) how fast is the immunization process compared to the viral infectious cycle; (iii) which other cellular pathways, if any, assist CRISPR immunization; (iv) how new spacer sequences are sampled from the invader's genome; and (v) how the immunization process affects the evolution of the host population; are not yet answered. The proposed studies will substantially advance our understanding of the molecular mechanisms underlying CRISPR-Cas immunization and the impact that these loci have on the ecology and evolution of prokaryotes organisms that harbor them. In addition, our experiments will require or allow us to engineer CRISPR-Cas systems that perform spacer acquisition with high frequency. Such systems will facilitate the development of technologies with applications that require the recording of specific cellular events into a specific genomic locus to enable researchers following long cellular histories. Thus the proposed studies could provide new ground to exploit CRISPR immunization for revolutionary biotechnological and/or therapeutic purposes.