Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR.
Nucleic Acids Res
; 50(11): e62, 2022 06 24.
Article
em En
| MEDLINE
| ID: mdl-35212386
ABSTRACT
CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy 'Ligation-Assisted Homologous Recombination' (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Sistemas CRISPR-Cas
/
Edição de Genes
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article