# Massively parallel reporter assays and genome editing of ENCODE predicted regulatory elements

> **NIH NIH UM1** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2021 · $1,409,478

## Abstract

Since its inception in 2003, the Encyclopedia of DNA Elements (ENCODE) Consortium has made remarkable
progress towards the identification of all functional elements in the human genome. However, major limitations
of the current catalog are that the vast majority of elements have not been functionally characterized, the impact
of genetic variation on their function is poorly defined, the precise levels of activation or repression that they
confer remain unmeasured, and the specific gene(s) that they regulate are not definitively known. To address
these gaps, we will implement ‘in genome’ massively parallel functional assays to characterize over 100,000
ENCODE-based candidate regulatory elements, to confirm and quantify their activities as well as to link many of
them to their target genes. In a systematic comparison of episomal vs. genomic massively parallel reporter
assays (MPRA), we show that episomal assays fail to accurately capture the full patterns of regulatory activity
that are observed in the context of chromatin. We therefore focus exclusively on methods that test candidate
regulatory elements in an integrated, ‘in genome’ context. First, using lentivirus-based massively parallel reporter
assays, we will characterize at least 100,000 ENCODE-based regulatory elements for their promoter/enhancer
activity while integrated into the genome (lentiMPRA; Aim 1a). Importantly, lentiMPRA can be carried out in
almost every cell type and leverages ongoing developments in lentivirus technology. Early results will be used
to iteratively develop models that make better selections for subsequent rounds of functional characterization.
Second, we will use CRISPR/Cas9 and multiplex homology directed repair to integrate a subset of candidate
enhancers to the 3’ UTR of transcriptionally inactive genes, allowing us to further validate and characterize their
ability to activate transcription in a natural genomic context (‘in genome’ STARR-seq; Aim 1b). Finally, we will
implement a new paradigm involving CRISPR/Cas9-based multiplex genome editing followed by RNAseq/
ATAC-seq molecular profiling to characterize a genome-wide subset of candidate regulatory elements in
their native genomic context for the functional consequences of mutations on them, while also determining the
target gene(s) that they regulate (massively parallel genome editing; Aim 2). Although we will initially focus our
efforts on K562 and HepG2 cells, we will also perform work in other cell lines as appropriate for the needs of the
ENCODE Consortium, with 25% of our capacity dedicated to a common set of elements. Combined with the
efforts of the other functional characterization centers, our work will provide unprecedented ‘in genome’ validation
and characterization of ENCODE-defined candidate regulatory elements, while also facilitating insights into our
understanding of the basic biology of gene regulation and how regulatory variants contribute to human disease
risk.

## Key facts

- **NIH application ID:** 10238522
- **Project number:** 3UM1HG009408-04S1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Nadav Ahituv
- **Activity code:** UM1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $1,409,478
- **Award type:** 3
- **Project period:** 2017-02-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10238522

## Citation

> US National Institutes of Health, RePORTER application 10238522, Massively parallel reporter assays and genome editing of ENCODE predicted regulatory elements (3UM1HG009408-04S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10238522. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*