lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Massively parallel reporter assays (MPRAs) are powerful tools for quantifying regulatory sequence function.
• Existing MPRA methods face limitations in transfecting certain cell types.
• A need exists for robust protocols and computational tools for MPRA data analysis.

Purpose of the Study:

• To present a detailed protocol for lentivirus-based MPRA (lentiMPRA).
• To introduce MPRAflow, a Nextflow-based computational pipeline for MPRA data analysis.
• To enable high-throughput functional characterization of regulatory elements, including in challenging cell types.

Main Methods:

• LentiMPRA involves cloning candidate regulatory sequences (CRSs) upstream of a reporter gene and a unique barcode.
• CRSs and barcodes are integrated into the genome via lentiviral transduction.
• RNA sequencing quantifies reporter transcription, normalized by DNA sequencing of integrated barcodes.

Main Results:

• The lentiMPRA protocol provides 'in-genome' readouts, facilitating MPRA in hard-to-transfect cells.
• The MPRAflow pipeline offers a user-friendly solution for quantifying CRS activity from MPRA data.
• The combined protocol and pipeline streamline the analysis of thousands of regulatory sequences.

Conclusions:

• LentiMPRA is a versatile method for assessing regulatory element function across diverse cell types.
• MPRAflow enhances the accessibility and efficiency of MPRA data analysis.
• This integrated approach accelerates the deciphering of regulatory codes and the development of genetic tools.