Establishment of a cloning-free CRISPR/Cas9 protocol to generate large deletions in the bovine MDBK cell line.
J Appl Genet
; 65(2): 399-402, 2024 May.
Article
em En
| MEDLINE
| ID: mdl-38418802
ABSTRACT
The CRISPR/Cas9 technique applied to modify the cattle genome has value in increasing animal health and welfare. Here, we established a simple, fast, and efficient cloning-free CRISPR/Cas9 protocol for large deletions of genomic loci in the frequently used model bovine MDBK cell line. The main advantages of our protocol are as follows (i) pre-screening of the sgRNA efficiency with a fast and simple cleavage assay, (ii) reliable detection of genomic edits primarily by PCR and confirmed by DNA sequencing, and (iii) single cell sorting with FACS providing specific genetic information from modified cells of interest. Therefore, our method could be successfully applied in different studies, including functional validation of any genetic or regulatory elements.
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Base de dados:
MEDLINE
Assunto principal:
Sistemas CRISPR-Cas
/
RNA Guia de Sistemas CRISPR-Cas
Idioma:
En
Ano de publicação:
2024
Tipo de documento:
Article