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

Autor: Kawamata M; Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. kawamata@bioreg.kyushu-u.ac.jp., Suzuki HI; Division of Molecular Oncology, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan. hisuzuki@med.nagoya-u.ac.jp.; Institute for Glyco-core Research (iGCORE), Nagoya University, Nagoya, Japan. hisuzuki@med.nagoya-u.ac.jp.; Center for One Medicine Innovative Translational Research, Gifu University Institute for Advanced Study, Gifu, Japan. hisuzuki@med.nagoya-u.ac.jp.; David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA. hisuzuki@med.nagoya-u.ac.jp., Kimura R; Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan., Suzuki A; Division of Organogenesis and Regeneration, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan. suzukicks@bioreg.kyushu-u.ac.jp.
Jazyk: angličtina
Zdroj: Nature biomedical engineering [Nat Biomed Eng] 2023 May; Vol. 7 (5), pp. 672-691. Date of Electronic Publication: 2023 Apr 10.
DOI: 10.1038/s41551-023-01011-7
Abstrakt: 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.
(© 2023. The Author(s).)
Databáze: MEDLINE
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