Application of a cost-effective DNA extraction protocol for screening transgenic and CRISPR-edited primary goat cells

• Main Authors: Louhanna Pinheiro Rodrigues Teixeira, Francisco Eder de Moura Lopes, André Saraiva Leão Marcelo Antunes, Matheus Soares Alves, André Marrocos Miranda, Saul Gaudencio Neto, Leonardo Tondello Martins, Ana Cristina de Oliveira Monteiro Moreira, Kaio Cesar Simiano Tavares, René Massimiliano Marsano
• Format: Article
• Language: English
• Published: Public Library of Science (PLoS) 2020-01-01
• Series: PLoS ONE
• Online Access: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500585/?tool=EBI

The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells—GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A—0%, protocol B—93%, protocol C—13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.