Physical stimuli-responsive CRISPR-Cas9 systems for spatiotemporally precise control of genome engineering

The Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR-Cas9) endonuclease system has revolutionized biology research by enabling precise, efficient, and versatile genome editing. However, achieving spatiotemporally controlled gene editing within specific organs, tissues, or cells remains a major challenge, as unregulated CRISPR-Cas9 activity can lead to severe off-target effects, hindering its clinical translation. To enhance the on-target precision and reduce the unwanted consequences of aberrant or premature CRISPR-Cas9 activation, various strategies have been developed to regulate its function at translational or post-translational stages using diverse external physicochemical stimuli. While chemical molecule-inducible CRISPR-Cas9 systems have demonstrated significant progress, most of them still suffer from inherent deficiencies, such as unsatisfactory spatiotemporal precision, irreversibility, pharmacokinetic dependence, internal disturbance, and safety concerns related to chemical inducers. By contrast, externally applied physical stimuli provide distinct advantages for triggering CRISPR-Cas9 activity, offering superior spatiotemporal precision, reversibility, and biocompatibility. These features significantly enhance the controllability, target specificity, and practical applicability of CRISPR-Cas9 systems across diverse biological settings. This review systematically explores recent advances in physical stimuli-responsive CRISPR-Cas9 platforms, detailing their design strategies, activation mechanisms, and proof-of-concept applications. Furthermore, we provide a comparative analysis of different stimulation strategies, highlighting their respective characteristics, current limitations, and future prospects. A discussion on the persistent bench-to-bedside gap is also included, aiming to guide future development toward clinically viable solutions.