Diverse Class 2 CRISPR-Cas Effector Proteins for Genome Engineering Applications

Abstract

CRISPR-Cas genome editing technologies have revolutionized modern molecular biology by making targeted DNA edits simple and scalable. These technologies are developed by domesticating naturally occurring microbial adaptive immune systems that display wide diversity of functionality for targeted nucleic acid cleavage. Several CRISPR-Cas single effector enzymes have been characterized and engineered for use in mammalian cells. The unique properties of the single effector enzymes can make a critical difference in experimental use or targeting specificity. This review describes known single effector enzymes and discusses their use in genome engineering applications.

Figure 1.

Graphic representing the diversity that enzyme choice may influence. Single effector Cas enzymes may be targeted to dsDNA or ssRNA, have different required protospacer flanking sequence requirements, may be delivered using different viral vectors, may be codon optimized and driven by promoters specific to function in different organisms, can be modified for higher specificity, and can be regulated.

Figure 2.

Basic structure and functions of Class 2 CRISPR-Cas Types. Cas9 from Streptococcus pyogenes represents Type II. Cas12a from Francisella novicida U112 represents Type V, and Cas13a from Leptotrichia shahii represents Type VI.

Figure 3.

Schematic representation of the unique Class 2 single effector Cas enzymes and their confirmed or predicted catalytic nuclease domains. Each Cas enzyme is grouped according to its CRISPR-Cas Type classification, and the number of orthologs is listed. The bright colors represent nuclease domains confirmed by crystal structure, and the faded colors represent computationally predicted nuclease domains. Several nuclease domains confirmed by crystal structure are not linear along the amino acid polypeptide chain and are split into sections as shown in the schematic. The split domains include RuvC-like in Cas9, Cas12a, and Cas12b and the first HEPN domain of Cas13a.