Practical application of massively parallel reporter assay in biotechnology and medicine

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Abstract

The development and viability of an organism relies on tissue-specific gene programs. The genome regulatory elements play a key role in the regulation of such programs, whereas its disfunction can lead to the establishment of various pathologies, including cancer, congenital disorders, and autoimmune diseases. The development of high-throughput approaches in genomics has led to the emergence of massively parallel reporter assays (MPRA), which enable genome-wide screening and functional verification of regulatory elements. Although MPRA was originally used for investigation of fundamental aspects of epigenetics, it also has a great potential for clinical and practical biotechnology. Currently, MPRA is used for validation of clinically significant mutations, identification of tissue-specific regulatory elements, identification of the favorable loci for transgene integration, as well as represents an essential tool for creating highly efficient expression systems, with a wide range of applications from protein production and design of novel therapeutic antibody super-producers to gene therapy. In this review, the basic principles and areas of practical application of high-throughput reporter assays will be discussed.

About the authors

Stanislav E. Romanov

Novosibirsk State University; Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences

Author for correspondence.
Email: romanov@mcb.nsc.ru
ORCID iD: 0000-0002-5989-5756
SPIN-code: 3387-6944
Scopus Author ID: 57201430841
ResearcherId: N-6935-2015
Russian Federation, Novosibirsk; 8/2, Acad. Lavrentiev Ave., Novosibirsk, 630090

Petr P. Laktionov

Novosibirsk State University; Institute of Molecular and Cellular Biology, Siberian Branch of the Russian Academy of Sciences

Email: laktionov@mcb.nsc.ru
ORCID iD: 0000-0003-2174-6496
SPIN-code: 7579-3460
Scopus Author ID: 57191597308
ResearcherId: N-7957-2015

PhD

Russian Federation, Novosibirsk; 8/2, Acad. Lavrentiev Ave., Novosibirsk, 630090

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2. Fig. 1. Strategies for massively parallel reporter assay: а — Principle of MPRA. Initially, a set of selected CREs is cloned into a pool of reporter constructs, each carrying a unique barcode. After transformation, RNA is extracted from the cells, and barcodes in the reporter transcripts is used as a measure of CRE activity; б — Schematic of the ReSE method for high-throughput detection of silencers (see explanations in the text); в — To study the post-transcriptional regulation of genes, the 5' or 3'- untranslated region (UTR) sequences or a transcription terminator with a polyadenylation signal are placed in the barcoded (BC) reporter construct. To investigate splicing, variants of introns flanked by different splice sites are placed in the barcoded construct. Disruption of splicing is accompanied by a shift in the content of the reporter proteins in the cells (GFP and mCherry). Next, cells can be categorized into groups based on the level of reporter protein activity, and barcode enrichment in each group can be analyzed.

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3. Fig. 2. Massively parallel reporter assays (MPRA) allow to confirms causative variants among the multiple candidates identified using GWAS (locus on the left). At the same time, causal mutations are often found within haplotypes containing multiple closely spaced polymorphisms (locus on the right). At such loci, GWAS is unable to identify the causal genetic variants, so the mechanism of disease development remains unclear. MPRA makes it possible to find polymorphisms in the haplotypes that disrupt gene regulation and identify the molecular basis of disease formation.

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