Analysis of compensatory mechanisms involved in response to genetic perturbation of drug transporters

A diverse repertoire of xenobiotic transporters from the ABC and SLC families are expressed in clinically relevant cell types. Structural and functional similarity of these transporters’ specificities is known to complicate efforts to perturb them. Given the therapeutic importance of these transporters, it is important to uncover the pathways that could lead to compensation for transporter loss‐of‐function. CRISPR/Cas9 based gene editing is extensively utilized for creating genetic lesions to analyze loss‐of‐function mutations. When applied in embryos a commonly observed outcome is genetic compensation. In the present study we take advantage of CRISPR/Cas9 based knockout of xenobiotic transporters in sea urchin embryos. Sea urchins express orthologs of several ABC transporters including P‐glycoprotein (ABCB1) and MRP1 (ABCC1). The fluorescent substrates and inhibitors of these transporters are also well‐defined providing simple system for analyzing changes in transporter activity in response to mutations. We have designed CRISPR single guide RNAs (sgRNAs) targeting orthologs of P‐glycoprotein (ABCB1, ABCB4), multidrug resistance proteins (ABCC1, ABCC4, ABCC5) and breast cancer resistance protein (ABCG2). Using a known CRISPR target in sea urchins, Nodal, we have successfully created small indels resulting in robust phenotypes. Using a bioinformatics tool called Inference of CRISPR Edits (ICE), we measured a cutting efficiency of 77.16% ±26.5 across four biological replicates. Knockdown of ABCB1a expression by antisense morpholino and inhibition of ABCB1a transporter activity with Valspodar (PSC833) was used to standardize the ABCB1a transporter activity in controls. We observed 2.5‐fold and 10‐fold increase in ABCB‐specific substrate, bodipy‐verapamil (b‐ver), in ABCB1a morphants and PSC inhibited embryos, respectively, while ABCC‐specific substrate, fluorescein diacetate (FDA) showed no change. Using microinjection of single as well as multiplex sgRNA, we now have successfully created cuts in ABCB1a loci resulting in small indels and large deletions, respectively. Currently, ABCB1a crispants are being analyzed for phenotypes using efflux assays. These crispants sometimes show increased accumulation of b‐ver, and strong efflux of FDA indicating compensation by ABCC1. We are also currently working to understand how variation in Cas9 cutting efficiency could relate to phenotypic variability. Recent studies have identified the role of Nonsense Mediated Decay (NMD) of mutant transcripts as one of the triggers for compensation which can be reversed by inhibiting the NMD pathway. As such, future work will focus on the role of mRNA stability and NMD on sensing of lesions in transporter loci. Results from this study could have impact on understanding the emergence of drug resistance and help design more effective ways to overcome it. Support or Funding Information Supported by R01ES027921 and R01ES030318 to Amro Hamdoun