Establishment of a cloning-free CRISPR/Cas9 protocol to generate large deletions in the bovine MDBK cell line

TL;DR

This paper introduces a streamlined CRISPR/Cas9 method to create large genomic deletions in bovine cells without cloning, offering a faster and more efficient approach for genetic studies.

Contribution

A novel cloning-free CRISPR/Cas9 protocol for large deletions in bovine MDBK cells is developed and validated.

Findings

A pre-screening cleavage assay efficiently identifies effective sgRNAs.

Genomic edits are reliably detected using PCR and confirmed by DNA sequencing.

Single-cell sorting via FACS enables precise genetic analysis of modified cells.

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.

The online version contains supplementary material available at 10.1007/s13353-024-00846-3.

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