6 In vitro validation of gene edited phenotypes using CRISPR-dCas9 transcriptional activators

Abstract

Despite the extensive advantages of gene-edited large animals for agriculture and biomedical purposes, they represent a large monetary and time investment due to high husbandry costs, long gestation lengths, and few offspring; that is, 9 months for one calf and almost 4 months for pigs. Even with known DNA sequences before somatic cell nuclear transfer (SCNT), inserted transgenes are often not expressed as expected. Therefore, there is a need to phenotypically validate the gene modifications in vitro before investing time and resources in the creation of a gene-edited large animal; however, many gene targets are tissue specific and not expressed in SCNT donor cells. In this work, we show that CRISPR-dCas9 transcriptional activators (TAs) can be used to validate functional transgene insertion in nonexpressing SCNT donor cells, in our case fetal fibroblasts. To demonstrate this concept, we first generated a DNA knockin of the H2B-GFP sequence into the porcine LGR5 locus. CRISPR/Cas9 nuclease was used to create a double-stranded break in the genomic DNA downstream of the LGR5 promoter. A homology-directed repair template plasmid containing H2B-GFP flanked by 1000 bp homology arms flanking the cut site was co-transfected with the Cas9 and gRNA, and cells were seeded at low density for colony outgrowth. Colonies were genotyped by PCR and sequencing to verify successful targeted transgene integration. To test whether TAs allow for in vitro validation of transgene expression, 5 × 10⁵ wildtype or gene-edited fibroblasts were nucleofected (Lonza) with 500 ng total of four gRNA plasmids (Addgene #43860) designed to target the 1-kb region upstream of the LGR5 transcriptional start site in combination with 500 ng VP64-dCas9 (Addgene #47107). Detection of green fluorescent protein (GFP) was analysed by fluorescent microscopy followed by flow cytometry; at least 30 000 events were recorded for each treatment (Cytoflex). Our results show that GFP was detected in on average 28.6% of the gene-edited cells transfected with LGR5 TAs but not detected in gene-edited cells that were not transfected with LGR5 TAs (0%) or in wild-type cells transfected with the LGR5 TAs (0%). The experiment was repeated three times. Next, to prove that our in vitro validation replicates the in vivo phenotype, the gene-edited colonies heterozygous for the insertion were used for SCNT to generate piglets. Epidermal cells, which contain a population of LGR5+ stem cells, were isolated from the skin and sorted for GFP expression. The RT-qPCR results from GFP+ or GFP− cells showed the presence of LGR5 transcripts in the GFP+ cells but not GFP− cells. In conclusion, TAs were necessary and sufficient to detect LGR5-promoter driven H2B-GFP expression in gene-edited fibroblasts in vitro, which faithfully recapitulates the in vivo phenotype of the gene-edited animal. Further preliminary data from our laboratory suggest that our novel method can be used to detect successful gene knockouts in addition to transgene knockins and can be used to validate phenotypic outcomes of DNA modifications before the generation of gene-edited animals.