Abstract

The archaeal and bacterial CRISPR -Cas systems employ small guide RNAs derived from foreign or self DNA for adaptive immunity against viruses and plasmids and apparently, in some case, also for regulation of gene expression. The RNA -guided Cas nucleases comprise the new generation of genome editing tools and are often claimed to have ushered a revolution in genetic engineering. The current classification of CRISPR -Cas systems includes 4 types and 11 subtypes of Class 1, with multisubunit effector complexes, and 2 types and 4 subtypes of Class 2, in which the effectors are single, multidomain proteins, exemplified by the experimentally characterized Cas9 and Cpf1. We developed a computational pipeline for comprehensive identification of CRISPR -Cas loci in genomic and metagenomic sequences, and employed it to search for novel Class 2 CRISPR -Cas systems. Four new subtypes of type V and a new type VI with 3 subtypes were discovered. The effectors of all type V systems contain the RuvC-like nuclease domain that apparently was derived from TnpB proteins encoded in bacterial and archaeal transposons but unlike the type II effectors, Cas9, lack the second, HNH nuclease domain. The type VI effector each contain two predicted HEPN R Nase domains. The crRNA processing was experimentally demonstrated for subtypes V-B and VI-A, and in the case of V-B, PAM -dependent interference was also identified. Further experimental characterization of these and other newly discovered CRISPR -Cas variants is underway. Sequence comparison and phylogenetic analysis indicate that Class 2 CRISPR -Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector genes acquired from distinct mobile elements. In addition to their importance in prokaryotic biology and utility as genome editing tools, the CRISPR -Cas systems shed light on general evolutionary principles. Utilization of transposon-encoded nucleases as “guns for hire” in defense systems appears to be one of such principles. The apparent continuum of evolutionary processes from Darwinian selection among random variants to the Lamarckian route of direct adaptation is another, higher plane generalization.