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

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.

Keywords: CRISPR/Cas9; Cattle; Deletion; MDBK; Regulatory elements.

Fig. 1

Cloning-free CRISPR/Cas9 approach for the generation of large genomic deletions using CRISPR/Cas9 and polymerase chain reaction (PCR) combined with gel electrophoresis. A Screening of sgRNA efficiency is performed by the cleavage assay on the region of interest amplified in two fragments. On the agarose gel, the overnight cleavage assay of the PCR products for fragment 1 and fragment 2 of the region of interest (Suppl. Table 2) using newly designed sgRNAs is shown. The PCR products before cleavage assay are marked by WT1 for fragment 1 and WT2 for fragment 2. The PCR products after the cleavage are marked by sgRNAs names (1A–1D for fragment 1 and 2A–2D for fragment 2). The most efficient gRNAs for each fragment (when less wild-type band is observed in the pattern after the cleavage and additional bands appeared after cleavage) for the further experiment are marked by an asterisk (*). B Transfection with ‘sandwich gRNA’ (combination of two selected sgRNAs). Below each sgRNA symbol, the PAM locations on chromosome 2 (BosTau9 version of the genome) are given. C Modifications in the genome after the transfection are detected primarily by PCR of the entire region of interest (different lengths of PCR products with and without deletion on agarose gel) and confirmed by DNA sequencing. The results of PCR amplification of the entire region of interest (using WTF1 and WTR1 primers) after the transfection of MDBK cells with the sandwich gRNAs are shown on the agarose gel. The PCR product for the wild-type (control) amplified using genomic DNA from MDBK cells before transfection is marked by ‘WT’. After transfection, the PCR product amplified using genomic DNA from MDBK cells where a possible deletion of the target element was detected in part of cells from the colony is marked by ‘D’. The band with deletion was marked by an asterisk (*). D FACS ensures accurate sorting of single cells (scattered by size) after transfection into 96-well plates, preventing the contamination by DNA from two single cells and providing specific genetic information from modified cell of interest only. On the agarose gel the wild-type (WT) and successful deletion (715 bp in length) of desired region D were shown