Effect of the Phosphoryl Guanidine Modification in Chimeric DNA-RNA crRNAs on the Activity of the CRISPR-Cas9 System .

Currently, the CRISPR-Cas9 system serves as a prevalent tool for genome editing and gene expression regulation. Its therapeutic application is limited by off-target effects that can affect genomic integrity through nonspecific, undesirable changes in the genome. Various strategies have been explored to mitigate the off-target effects.

Expression and Functional Analysis of the Compact Thermophilic Cas9 Nuclease.

Research on Cas9 nucleases from different organisms holds great promise for advancing genome engineering and gene therapy tools, as it could provide novel structural insights into CRISPR editing mechanisms, expanding its application area in biology and medicine. The subclass of thermophilic Cas9 nucleases is actively expanding due to the advances in genome sequencing allowing for the meticulous examination of various microorganisms' genomes in search of the novel CRISPR systems. The most prominent thermophilic Cas9 effectors known to date are GeoCas9, ThermoCas9, IgnaviCas9, AceCas9, and others.

Influence of N1-Methylpseudouridine in Guide RNAs on CRISPR/Cas9 Activity.

At present, there are many strategies to improve the activity of CRISPR/Cas9. A well-known and effective approach is guide RNA modification. Many chemical guide RNA modifications have been studied, whereas naturally occurring RNA modifications are largely unexplored.

Natural Nucleoside Modifications in Guide RNAs Can Modulate the Activity of the CRISPR-Cas9 System .

At the present time, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system has been widely adopted as an efficient genomic editing tool. However, there are some actual problems such as the off-target effects, cytotoxicity, and immunogenicity. The incorporation of modifications into guide RNAs permits enhancing both the efficiency and the specificity of the CRISPR-Cas9 system.

Thermodynamic Swings: How Ideal Complex of Cas9-RNA/DNA Forms.

Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR-Cas9 tool.