Quantitative spatial and temporal assessment of regulatory element activity in zebrafish

Abstract

Mutations or genetic variation in noncoding regions of the genome harbouring cis-regulatory elements (CREs), or enhancers, have been widely implicated in human disease and disease risk. However, our ability to assay the impact of these DNA sequence changes on enhancer activity is currently very limited because of the need to assay these elements in an appropriate biological context. Here, we describe a method for simultaneous quantitative assessment of the spatial and temporal activity of wild-type and disease-associated mutant human CRE alleles using live imaging in zebrafish embryonic development. We generated transgenic lines harbouring a dual-CRE dual-reporter cassette in a pre-defined neutral docking site in the zebrafish genome. The activity of each CRE allele is reported via expression of a specific fluorescent reporter, allowing simultaneous visualisation of where and when in development the wild-type allele is active and how this activity is altered by mutation.

Heterogeneity and genomic loci of ubiquitous transgenic Cre reporter lines in zebrafish

“…Previously generated transgene integrations for repeated insertion on the phiC31 recombinase system have shown weak to medium expression levels, have not been validated for lox-based recombination, or have not been maintained beyond proof-of-principle. 1,[53][54][55][56][57][58] Consequently, versatile and validated safe harbor sites are currently missing in zebrafish, rendering Tol2-or ISce-I-based random integration and subsequent screening for functional single-copy transgenes labor-intensive. Recent work has reported the Tol2-based generation of a phiC31-targetable attB integration site called SHH-SBE2 that is suitable for generegulatory element analysis through reproducible transgenesis into the same locus 57 while promising, if SHH-SBE2 shows long-term stability and is amenable to loxbased Switch reporters warrants further investigation.…”

“…1,[53][54][55][56][57][58] Consequently, versatile and validated safe harbor sites are currently missing in zebrafish, rendering Tol2-or ISce-I-based random integration and subsequent screening for functional single-copy transgenes labor-intensive. Recent work has reported the Tol2-based generation of a phiC31-targetable attB integration site called SHH-SBE2 that is suitable for generegulatory element analysis through reproducible transgenesis into the same locus 57 while promising, if SHH-SBE2 shows long-term stability and is amenable to loxbased Switch reporters warrants further investigation. Retained switching efficiency over generations is a highly desirable feature of lox-based Switch reporter transgenics.…”

“…So‐called safe harbor sites to integrate transgenes provide a key tool for reproducible transgene deployment in any given model. Previously generated transgene integrations for repeated insertion based on the phiC31 recombinase system have shown weak to medium expression levels, have not been validated for lox ‐based recombination, or have not been maintained beyond proof‐of‐principle 1,53‐58 . Consequently, versatile and validated safe harbor sites are currently missing in zebrafish, rendering Tol2‐or ISce‐I‐based random integration and subsequent screening for functional single‐copy transgenes labor‐intensive.…”

Functional Validation of Osteoporosis Genetic Findings Using Small Fish Models

“…Testing enhancer activity and comparing with the impact of single nucleotide changes within these sequences are already possible in zebrafish. While most of the designed engineering approaches to test enhancers in zebrafish rely on variable genomic site integration, recent improvements in the technology permit to control genomic integration, reducing noise and increasing reproducibility [ 194 ]. Given the transparency of zebrafish, the activity of enhancer elements can be monitored in a temporospatial way, at least during development.…”

Instantaneous visual genotyping and facile site-specific transgenesis via CRISPR-Cas9 and phiC31 integrase

“…Hence, the development of this technique may widen the use of the zebrafish as a platform to rapidly assess the functions of genetic variants in the coding and non-coding regions identified in genome-wide association studies (GWAS) (Edwards et al, 2013; Gusev et al, 2018; Tucker et al, 2017). Indeed, the phiC31 transgenesis method via several Tol2-generated attP lines has been shown to be superior to the Tol2 transgenesis method when evaluating human cis-regulatory elements in zebrafish (Bhatia et al, 2021; Roberts et al ., 2014). The landing sites in the integration recipient lines used in previous studies have been mapped to both intergenic and intragenic regions (Bhatia et al ., 2021; Mosimann et al ., 2013b; Roberts et al ., 2014).…”