Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs

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  • 川又, 理樹
    九州大学大学院生体防御医学研究所細胞機能制御学部門器官発生再生学分野
  • 鈴木, 洋
    名古屋大学大学院医学系研究科・医学部医学科 名古屋大学統合糖鎖研究拠点 岐阜大学One Medicine 創薬シーズ開発・育成研究教育拠点
  • キムラ, リョウタ
    九州大学大学院生体防御医学研究所細胞機能制御学部門器官発生再生学分野
  • 鈴木, 淳史
    九州大学大学院生体防御医学研究所細胞機能制御学部門器官発生再生学分野

説明

The precise regulation of the activity of Cas9 is crucial for safe and efficient editing. Here we show that the genome-editing activity of Cas9 can be constrained by the addition of cytosine stretches to the 5′-end of conventional single-guide RNAs (sgRNAs). Such a ‘safeguard sgRNA’ strategy, which is compatible with Cas12a and with systems for gene activation and interference via CRISPR (clustered regularly interspaced short palindromic repeats), leads to the length-dependent inhibition of the formation of functional Cas9 complexes. Short cytosine extensions reduced p53 activation and cytotoxicity in human pluripotent stem cells, and enhanced homology-directed repair while maintaining bi-allelic editing. Longer extensions further decreased on-target activity yet improved the specificity and precision of mono-allelic editing. By monitoring indels through a fluorescence-based allele-specific system and computational simulations, we identified optimal windows of Cas9 activity for a number of genome-editing applications, including bi-allelic and mono-allelic editing, and the generation and correction of disease-associated single-nucleotide substitutions via homology-directed repair. The safeguard-sgRNA strategy may improve the safety and applicability of genome editing.

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