Unbiased whole genome detection of ultrarare off-target mutations in genome-edited cell populations by HiFi sequencing.
Environ Mol Mutagen
; 64(7): 374-381, 2023 08.
Article
em En
| MEDLINE
| ID: mdl-37488781
ABSTRACT
DNA base editors (BEs) composed of a nuclease-deficient Cas9 fused to a DNA-modifying enzyme can achieve on-target mutagenesis without creating double-strand DNA breaks (DSBs). As a result, BEs generate far less DNA damage than traditional nuclease-proficient Cas9 systems, which do rely on the creation of DSBs to achieve on-target mutagenesis. The inability of BEs to create DSBs makes the detection of their undesired off-target effects very difficult. PacBio HiFi sequencing can efficiently detect ultrarare mutations resulting from chemical mutagenesis in whole genomes with a sensitivity ~1 × 10-8 mutations per base pair. In this proof-of-principle study, we evaluated whether this technique could also detect the on- and off-target mutations generated by a cytosine-to-thymine (C>T) BE targeting the LacZ gene in Escherichia coli (E. coli). HiFi sequencing detected on-target mutant allele fractions ranging from ~7% to ~63%, depending on the single-guide RNA (sgRNA) used, while no on-target mutations were detected in controls lacking the BE. The presence of the BE resulted in a ~3-fold increase in mutation frequencies compared to controls lacking the BE, irrespective of the sgRNA used. These increases were mostly composed of CG>TA substitutions distributed throughout the genome. Our results demonstrate that HiFi sequencing can efficiently identify on- and off-target mutations in cell populations that have undergone genome editing.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Sistemas CRISPR-Cas
/
RNA Guia de Sistemas CRISPR-Cas
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article