NOVEL CRISPR-CAS SYSTEMS FROM UNCULTIVATED MICROBES

David Burstein ¹ Lucas B. Harrington ² Janice S. Chen ² Jillian F. Banfield ^3,5 Jennifer A. Doudna ^2,4,5,6

¹School of Molecular Cell Biology and Biotechnology, Tel Aviv University, Tel Aviv, Israel
²Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, CA, USA
³Department of Earth and Planetary Sciences, University of California, Berkeley, Berkeley, CA, USA
⁴Department of Chemistry, University of California Berkeley, Berkeley, CA, USA
⁵Innovative Genomics Institute, University of California Berkeley, Berkeley, CA, USA
⁶Howard Hughes Medical Institute, University of California Berkeley, Berkeley, CA, USA

Current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth’s ecosystems, is based primarily on cultivated organisms. We applied a cultivation-independent approach to seek new CRISPR-Cas systems that will contribute to our understanding of the arms race between viruses and their microbial hosts, and potentially expand the genome editing toolbox. Using genome-resolved metagenomics, we identify a number of novel CRISPR–Cas systems, including the first reported Cas9 in the archaeal domain of life. In bacteria, we discovered previously unknown families of CRISPR-Cas systems, which are among the most compact systems known to date. Notably, all required functional components were identified by environmental metagenomics and metatranscriptomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes and test the function of microbial systems that will expand the repertoire of microbial genes utilized in biotechnology and elucidate the evolutionary processes that led to the rise of these systems.