In this study, we present a protocol to obtain deletions of specific genomic regions in MDBK cells using CRISPR/Cas9 and PCR combined with gel electrophoresis as a quick and reliable approach to analyse the efficiency of designed sgRNAs.

Only a few studies have generated CRISPR-edited cell lines using MDBK cells. Chen et al. (2021) conducted transduction with lentiviral vectors encoding the sgRNAs under the control of a U6 promoter and Cas9 to produce novel MDBK CD46 knockout clones, selected by interaction with the anti-bovine CD46 monoclonal antibody CA26. Other CD46 knockout cells have been obtained by Szillat et al. (2020) using a CRISPR/Cas9 ribonucleoproteins (RNPs)-mediated approach. However, to our knowledge, a comprehensive protocol for CRISPR-mediated large genomic deletions using RNPs has not been reported.

Here, we described a swift and easy protocol for successfully designing and testing sgRNAs and the subsequent generation of a large deletion in the desired genome location. To choose and test the best-fitting crRNAs forming ‘sandwich gRNA’, we suggest designing and ordering several crRNAs in one batch. In our experiments, we used the combination of the two most efficient crRNAs (‘sandwich gRNA’) that cut the genomic DNA in two sites, at the beginning and the end of the desired region in the genome. In our method, several crRNAs were tested on MDBK DNA as a preliminary step using PCR and subsequent agarose gel visualization. The amplification of the wild-type region of interest was successful, and three amplicons were obtained—for fragment 1, fragment 2, and the entire wild-type region (Suppl. Table 1). The efficiency of the tested sgRNAs (1A–1D and 2A–2D) for fragment 1 and fragment 2 is presented in Fig. 1A. The crRNA-1B and crRNA-2D were chosen based on their cleavage efficiency (when, after cleavage, a less wild-type band is observed in the pattern together with additional bands present). After transfection of MDBK cells using the sandwich gRNAs (1B2D), genomic DNA was extracted, and the deletion was confirmed by PCR of the entire wild-type region, using WTF1 and WTR1 primers (Fig. 1B, C; Suppl. Table 1). The transfected cells with confirmed deletion (Fig. 1C) were separated by FACS into four 96-well plates and checked after 3 days.

In our protocol, we used 72-h incubation during the transfection process. Shorter incubation time (48 h) showed no significant difference in the transfection success (data not shown). The MDBK cells proliferated fast in our culture condition. Therefore, single-cell colony identification was possible after 3 days. In the case of other cell lines, it is important to adjust the protocol to the proliferation time.

The success rate (assessed by observation under the optical microscope) of monoclonal colony formation by FACS was 25.3% (97/384). Screening of all 97 potentially single-cell colonies, was performed after one additional week. Cells were trypsinized and diluted in 100 µl, of which 50 µl was used for PCR screening and visualization on agarose gel. Using this PCR screen, we can predict whether the obtained clone possesses homozygous or heterozygous deletions of the targeted region. Furthermore, this approach allows us to exclude the possible contamination of non-single cell-derived clones.

Only four clones with a clear single band of approximately 800 bp in length identified on the agarose gel (Fig. 1D), indicating successful deletion of the region of interest in a homozygote manner, were chosen for DNA sequencing. The DNA sequence was checked for all four clones and confirmed the targeted deletion (Suppl. Mats 1).