Fourth, in order to determine the phenotypic effect of an enhancer variant, it is essential that an enhancer variant is demonstrated to influence the levels of its target transcript. The target may be a gene, or could alternatively be a noncoding RNA. However, enhancers frequently regulate multiple genes. Even if the levels of a given transcript correlate with the genotype of an enhancer risk variant, it does not necessarily mean that the correlated gene is causal. Functional assays, and ultimately in vivo models, are needed to verify that the gene is directly involved in the development of the disease. CRISPR/Cas9 technology would enable such studies by altering single SNPs in the genome of a model organism while maintaining the native genomic context of the variant. Alternatively, single-site integration of the risk or non-risk alleles into a model organism, as utilized for the enhancer variant associated with blond hair color [33], could be employed. Although CRISPR/Cas9 can be utilized to demonstrate the functional impact of a given variant, the complex phenotypes of many diseases are not easily modeled in vitro and thus the determination of causality will often not be trivial.
