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

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  • Kawamata, Masaki
    Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University
  • Suzuki, Hiroshi I.
    Division of Molecular Oncology, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine Institute for Glyco-core Research (iGCORE), Nagoya University Center for One Medicine Innovative Translational Research, Gifu University Institute for Advanced Study David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology
  • Kimura, Ryota
    Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University
  • Suzuki, Atsushi
    Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University

Description

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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