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

Abstract

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.

Targeted Gene Delivery: Where to Land

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The Application of the CRISPR/Cas9 System in the Treatment of Hepatitis B Liver Cancer

“…Among them, nontarget effects caused by the overexpression of the Cas9 protein will be the most difficult to overcome in research. 88 In a study, scientists conducted a high-throughput evaluation of the activity of CRISPR/Cas9 to examine which small-molecule drugs can effectively inhibit its activity. To this end, they built 2 systems.…”

Genetic analyses of root traits: Implications for environmental adaptation and new variety development: A review

“…The CRISPR/Cas9 is a precise technique used to insert or knock out specific genes in organisms, including plants (Liu & Qin, 2021). However, off-target gene insertions or deletions have been reported when using CRISPR (Huang et al, 2021) requiring appropriate primers and functional genes (Wang et al, 2020). Off-target effects of CRISPR/Cas9 can occur in crop species such as maize, soybean, rice and wheat that possess several duplicated genes (Li et al, 2016).…”