Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox

CRISPRsystems mediate adaptive immunity in bacteria and archaeathrough diverse effector mechanisms and have been repurposed for versatileapplications in therapeutics and diagnostics thanks to their facilereprogramming with RNA guides. RNA-guided CRISPR-Cas targeting andinterference are mediated by effectors that are either componentsof multisubunit complexes in class 1 systems or multidomain single-effectorproteins in class 2. The compact class 2 CRISPR systems have beenbroadly adopted for multiple applications, especially genome editing,leading to a transformation of the molecular biology and biotechnologytoolkit. The diversity of class 2 effector enzymes, initially limitedto the Cas9 nuclease, was substantially expanded via computationalgenome and metagenome mining to include numerous variants of Cas12and Cas13, providing substrates for the development of versatile,orthogonal molecular tools. Characterization of these diverse CRISPReffectors uncovered many new features, including distinct protospaceradjacent motifs (PAMs) that expand the targeting space, improved editingspecificity, RNA rather than DNA targeting, smaller crRNAs, staggeredand blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage,etc. These unique properties enabled multiple applications, such asharnessing the promiscuous RNase activity of the type VI effector,Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systemshave been adopted for genome editing, as well, despite the challengeof expressing and delivering the multiprotein class 1 effectors. Therich diversity of CRISPR enzymes led to rapid maturation of the genomeediting toolbox, with capabilities such as gene knockout, base editing,prime editing, gene insertion, DNA imaging, epigenetic modulation,transcriptional modulation, and RNA editing. Combined with rationaldesign and engineering of the effector proteins and associated RNAs,the natural diversity of CRISPR and related bacterial RNA-guided systemsprovides a vast resource for expanding the repertoire of tools formolecular biology and biotechnology.