Unity among the diverse RNA-guided CRISPR-Cas interference mechanisms.

CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are adaptive immune systems that protect bacteria and archaea from invading mobile genetic elements (MGEs). The Cas protein-CRISPR RNA (crRNA) complex uses complementarity of the crRNA "guide" region to specifically recognize the invader genome. CRISPR effectors that perform targeted destruction of the foreign genome have emerged independently as multi-subunit protein complexes (Class 1 systems) and as single multi-domain proteins (Class 2). These different CRISPR-Cas systems can cleave RNA, DNA, and protein in an RNA-guided manner to eliminate the invader, and in some cases, they initiate programmed cell death/dormancy. The versatile mechanisms of the different CRISPR-Cas systems to target and destroy nucleic acids have been adapted to develop various programmable-RNA-guided tools and have revolutionized the development of fast, accurate, and accessible genomic applications. In this review, we present the structure and interference mechanisms of different CRISPR-Cas systems and an analysis of their unified features. The three types of Class 1 systems (I, III, and IV) have a conserved right-handed helical filamentous structure that provides a backbone for sequence-specific targeting while using unique proteins with distinct mechanisms to destroy the invader. Similarly, all three Class 2 types (II, V, and VI) have a bilobed architecture that binds the RNA-DNA/RNA hybrid and uses different nuclease domains to cleave invading MGEs. Additionally, we highlight the mechanistic similarities of CRISPR-Cas enzymes with other RNA cleaving enzymes and briefly present the evolutionary routes of the different CRISPR-Cas systems.

Rational Engineering of CRISPR-Cas9 Nuclease to Attenuate Position-Dependent Off-Target Effects.

The RNA-guided Cas9 nuclease from Streptococcus pyogenes has become an important gene-editing tool. However, its intrinsic off-target activity is a major challenge for biomedical applications. Distinct from some reported engineering strategies that specifically target a single domain, we rationally introduced multiple amino acid substitutions across multiple domains in the enzyme to create potential high-fidelity variants, considering the Cas9 specificity is synergistically determined by various domains. We also exploited our previously derived atomic model of activated Cas9 complex structure for guiding new modifications. This approach has led to the identification of the HSC1.2 Cas9 variant with enhanced specificity for DNA cleavage. While the enhanced specificity associated with the HSC1.2 variant appeared to be position-dependent in the in vitro cleavage assays, the frequency of off-target DNA editing with this Cas9 variant is much less than that of the wild-type Cas9 in human cells. The potential mechanisms causing the observed position-dependent effect were investigated through molecular dynamics simulation. Our discoveries establish a solid foundation for leveraging structural and dynamic information to develop Cas9-like enzymes with high specificity in gene editing.

The Whole Is Bigger than the Sum of Its Parts: Drug Transport in the Context of Two Membranes with Active Efflux.

Cell envelope plays a dual role in the life of bacteria by simultaneously protecting it from a hostile environment and facilitating access to beneficial molecules. At the heart of this ability lie the restrictive properties of the cellular membrane augmented by efflux transporters, which preclude intracellular penetration of most molecules except with the help of specialized uptake mediators. Recently, kinetic properties of the cell envelope came into focus driven on one hand by the urgent need in new antibiotics and, on the other hand, by experimental and theoretical advances in studies of transmembrane transport. A notable result from these studies is the development of a kinetic formalism that integrates the Michaelis-Menten behavior of individual transporters with transmembrane diffusion and offers a quantitative basis for the analysis of intracellular penetration of bioactive compounds. This review surveys key experimental and computational approaches to the investigation of transport by individual translocators and in whole cells, summarizes key findings from these studies and outlines implications for antibiotic discovery. Special emphasis is placed on Gram-negative bacteria, whose envelope contains two separate membranes. This feature sets these organisms apart from Gram-positive bacteria and eukaryotic cells by providing them with full benefits of the synergy between slow transmembrane diffusion and active efflux.