Chemically composed Cas9 (ChemComCas9) for spatio-temporal regulation and precision genome engineering applications.

Chemically composed Cas9 (ChemComCas9) has emerged as a highly versatile CRISPR enzyme, offering tunable, on-demand, and targeted control over genome engineering functions. Despite its programmable, sequence-specific activity, uncontrolled or prolonged exposure to Cas9 can lead to severe genotoxicity, restricting its enormous clinical potential. To circumvent its non-specific and uncontrolled activities, Cas9 has been chemically modified for spatio-temporal and precision control. For developing such ChemComCas9, the WT Cas9 has been chemically modified using either its surface-exposed natural cysteine or by the installation of additional cysteine residues, or an unnatural amino acid has been incorporated for chemical modification. Such strategies include the incorporation of peptides, polymers, antibodies, stimuli-responsive moieties, and biorthogonal conjugation techniques that allow external control over Cas9 activation, localization, and activity. In addition, various short peptides have been either genetically fused or chemically conjugated for spatiotemporal regulation and probing. These approaches enable conditional activation of Cas9 in response to light, small molecules, or intracellular triggers, thereby minimizing off-target effects and increasing editing efficiency. In this review, we discuss a systematic evolution of chemically composed Cas9 (ChemComCas9) for precision genome engineering and gene therapy applications.