Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9.

CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions.

Strategies for Optimization of the Clustered Regularly Interspaced Short Palindromic Repeat-Based Genome Editing System for Enhanced Editing Specificity.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is inarguably the most valuable gene editing tool ever discovered. Currently, three classes of CRISPR-based genome editing systems have been developed for gene editing, including CRISPR/CRISPR associate system (Cas) nucleases, base editors, and prime editors. Ever-evolving CRISPR technology plays an important role in medicine; however, the biggest obstacle to its use in clinical practice is the induction of off-target effects (OTEs) during targeted editing. Therefore, continuous improvement and optimization of the CRISPR system for reduction of OTEs is a major focus in the field of CRISPR research. This review aims to provide a comprehensive guide for optimization of the CRISPR-based genome editing system.

Development of a Self-Restricting CRISPR-Cas9 System to Reduce Off-Target Effects.

Development of the CRISPR-Cas9 gene-editing system has given rise to a new era of gene editing with wide applications in biology, medicine, agriculture, and other fields. However, the overexpression of Cas9 nuclease causes off-target effects and may trigger an immune response in vivo. Therefore, we constructed a self-restricting CRISPR-Cas9 system, where the target gene sequence corresponding to the guide RNA (gRNA) is inserted on either end of the Cas9 promoter. When double-strand breaks (DSBs) are induced in the target gene sequence, the Cas9 promoter is cut off and transcription ceases. With this system, expression of Cas9 protein at 60 h after transfection is only 10% that of the wild-type system, with about 70% promoter deletion efficiency. The target site editing efficiency and homologous recombination efficiency of the self-restricting system remain at about 50% and 30%, respectively, while the frequency of off-target indel formation decreased by 76.7%. Further, the number of indel types was also reduced from 13 to 2. Because this system does not include additional gRNA sequences, the possibility of introducing new off-target mutations is decreased. Importantly, this system is composed of a single plasmid, which could potentially be easily introduced in vivo using a viral vector or nanoparticles.