Improving CRISPR-Cas technology for therapeutic applications

Abstract

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CRISPR-Cas technology has rapidly become a popular and rather powerful tool of genome manipulation. It is widely used not only for scientific research but for the development of new therapeutic approaches. Applications of CRISPR-CAS system are not limited by genome editing, they include genetic screens, modulation of gene expression either by attracting RNA polymerase or by blocking its binding to DNA, induction of histone or genome methylation and silencing, incorporation of specific sequences into the target site in the genome. Whatever modification of the CRISPR-Cas technology is used, it relies deeply on the target recognition by CRISPR-Cas complex, based on the complementarity of a rather short oligonucleotide sequence. The efficiency and specificity of this binding of spacer part of the guide RNA to DNA are especially important for therapeutic applications of CRISPR-Cas system. However, its selectivity is still insufficient for routine in vivo use. Uncontrolled insertion or removal of genes and their fragments in the "hot spots” of the genome can lead to oncotransformation of cells after genome editing ex vivo or in vivo. Another important problem for therapy is targeted delivery of the preformed RNA-protein complex or insufficient level of nuclear localization of synthetic guide RNAs if they are not part of the protein. Here we use combinations of known and novel chemical RNA modifications to improve the characteristics of precise genome editing for its potential applications in biomedicine. These novel modifications stabilize RNA in vivo and are less toxic then phosphorothioates which are widely used to prevent RNA degradation of synthetic RNA molecules in vivo. We identify patterns of RNA modifications which can further improve therapeutic efficacy of CRISPR-Cas system without decrease of genome editing efficiency and compare them to the known gRNA modifications patterns. We also analyze the effects of these RNA modifications on the protein-RNA complex activity and discuss possible alterations in the structure of Cas9 effector complex caused by these modifications.