Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes. We developed a high-throughput pipeline that enabled us to predict tracrRNA sequences, to design single guide RNAs, and to demonstrate nuclease activityin vitrofor 41 newly described subgroups. Active systems represent an extensive diversity of protein sequences and guide RNA structures and require diverse protospacer adjacent motifs (PAMs) that collectively expand the known targeting capability of current systems. Several nucleases showed activity levels comparable to or significantly higher than SpCas9, despite being smaller in size. In addition, top systems exhibited low levels of off-target editing in mammalian cells, and PAM-interacting domain engineered chimeras further expanded their targetability. These newly discovered nucleases are attractive enzymes for translation into many applications, including therapeutics.