In:
Nature Biomedical Engineering, Springer Science and Business Media LLC, Vol. 7, No. 5 ( 2023-04-10), p. 672-691
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
2157-846X
DOI:
10.1038/s41551-023-01011-7
Language:
English
Publisher:
Springer Science and Business Media LLC
Publication Date:
2023
detail.hit.zdb_id:
2878897-7
