UC Berkeley

Bacterial and archaeal genomes are under constant threat by genetic invaders. The need to maintain genomic and cellular integrity has driven the evolution of numerous and diverse genome defense systems. A common theme in prokaryotic defense strategies is interference of foreign DNA and RNA on the sequence level. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems confer adaptive immunity to previously encountered genetic invaders. Guided by short RNAs, the main effectors of CRISPR-Cas systems are sequence specific nucleases that catalyze degradation of exogenous nucleic acids. At the center of a similar method of genome defense to CRISPR-Cas systems, but operating through non-homologous proteins and pathways, prokaryotic Argonaute proteins (pAgos) have been proposed as sequence specific defense systems. However, our mechanistic knowledge of both CRISPR-Cas and pAgo systems stems from a small subset of the total genetic diversity of these systems. Here, we address this limited understanding through analysis of new CRISPR-Cas and pAgo systems, as well as describe novel activity for previously identified members.

CRISPR-Cas9 has rapidly been adopted as a programmable platform for sequence-specific DNA targeting with endless applications in gene editing, genome-wide screening, and transcriptional control. Current applications draw upon the biochemical activities of a few common Cas9 enzymes. The study of diverse homologs has potential to yield novel Cas9 proteins with desired traits such as increased efficiency or specificity. Surveying a vast metagenomic database, we report the first Cas9 from archaea, expanding the occurrence of CRISPR-Cas9 systems to a new domain of life.

DNA targeting is a hallmark of CRISPR-Cas9 systems. Engineering SpyCas9 to bind and target RNA has been difficult and suffers from reduced efficiency. We sought to identify Cas9 homologs with a natural ability to target RNA molecules. Using in vitro purification and biochemical assays, we discovered Cas9 enzymes that efficiently cleave RNA. Furthermore, we show that this activity can be harnessed to reduce phage infection and mediate gene repression in vivo, expanding the toolkit of CRISPR-Cas nucleases.

Analogous to CRISPR-Cas systems, Argonaute proteins are well known, RNA-guided nucleases that operate in eukaryotic RNA-interference. Motivated by initial observations that Argonaute homologs in prokaryotes constitute a nucleic acid-guided genome defense system, we studied the physiological role and biochemical activities of a novel clade of pAgos. We offer the first experimental evidence of complex formation between natural, two-piece Argonaute proteins. Preliminary in vivo observations implicate this split-pAgo in maintaining motility under conditions induced by introduction of an exogenous plasmid. Together with our studies on CRISPR-Cas systems, our work highlights unexpected functional diversity across divergent nucleic acid-guided genome defense systems.