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
in 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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
CRISPR-Cas Systems
/
RNA, Guide, CRISPR-Cas Systems
Limits:
Animals
Language:
En
Journal:
J Appl Genet
Journal subject:
GENETICA
Year:
2024
Document type:
Article
Affiliation country:
Poland