In the vast non-coding regions of our DNA, a revolutionary tool is uncovering hidden switches that control our biology.
For decades, the spotlight in genetics has shone on protein-coding genes, which make up a mere 1-2% of the human genome. The remaining 98%, once dismissed as "junk DNA," is now recognized as a critical control layer filled with switches that dictate when and where genes are activated. Until recently, mapping these switches was slow and laborious. The Molecular Chipper technology has emerged as a powerful solution, using CRISPR to systematically expose these hidden regulators and illuminate the dark matter of our genome2 4 6 .
Protein-coding portion of human genome
Non-coding "junk DNA" with regulatory functions
Base pairs length of typical regulatory elements
The non-coding genome is populated by diverse regulatory elements6 :
Short DNA regions that can boost gene expression, sometimes located far from the genes they control.
Elements that suppress or reduce gene activity.
Genomic "buffer" zones that can block enhancers from acting on the wrong genes.
Regions that initiate the transcription of a gene.
Identifying these elements is one challenge; proving their function is another. Traditional methods relied on correlative evidence like epigenetic marks or evolutionary conservation, but these approaches couldn't demonstrate causal relationships2 . CRISPR-Cas9 provided a revolutionary tool for making precise cuts in DNA, but when it came to studying non-coding regions, a significant limitation emerged: most regulatory elements are 50-200 base pairs long, while a single CRISPR cut typically creates only small insertions or deletions (indels). Destroying an entire functional element often required more extensive DNA removal6 .
The Molecular Chipper technology, introduced in a 2016 Nature Communications paper, addresses this fundamental challenge by generating exceptionally dense single-guide RNA (sgRNA) libraries specifically for interrogating non-coding regions4 .
The innovative approach combines random fragmentation with enzymatic processing to create comprehensive coverage of targeted genomic areas4 :
Researchers begin with DNA encompassing the non-coding region of interest.
This DNA is randomly broken into smaller pieces.
A type III restriction enzyme is used to process these fragments.
The final output is a dense sgRNA library with extensive coverage.
This method is inexpensive, flexible, and easy to implement, allowing scientists to create custom libraries for any genomic region without prior assumptions about where functional elements might be located4 .
| Approach | Library Design | Throughput | Key Advantage |
|---|---|---|---|
| Individual sgRNA | Targeted to specific sites | Low | Simple for narrow, pre-defined targets |
| Dual-sgRNA/CRISPR Del | Paired guides for deletion | Low-to-High | Removes large regions systematically |
| Molecular Chipper | Random fragmentation-based | High | Dense, unbiased coverage of any region |
| dCas9 Screening | Targeted to specific sites | High | Modulates activity without cutting DNA |
The study that introduced Molecular Chipper provided a compelling demonstration of its power by investigating the biogenesis of miR-142, a microRNA crucial for immune regulation4 .
The researchers applied the Molecular Chipper technology to 17 microRNAs and their flanking regions, generating a dense sgRNA library representing these areas.
They introduced this library, along with the Cas9 nuclease, into cells containing a sensor for miR-142 activity.
Cells were screened for changes in miR-142 function. When a CRISPR cut disrupted a critical regulatory domain, the sensor would signal a loss of activity.
Genomic DNA from the screened cells was sequenced to identify which sgRNAs were enriched or depleted, pinpointing the exact DNA sequences essential for miR-142 function.
The experiment successfully identified two key types of functional regions4 :
This was a significant discovery because it revealed that the regulation of microRNA biogenesis extends beyond the core sequence itself into flanking regions that were previously uncharacterized. The Molecular Chipper technology enabled this discovery without any prior bias about where these regulatory domains might be located.
| Genomic Region | Known Function | Newly Discovered Role |
|---|---|---|
| pre-miR-142 sequence | Core component for mature miR-142 production | Confirmed as essential |
| Cis-regulatory Domain 1 | Previously uncharacterized | Critical for miR-142 biogenesis |
| Cis-regulatory Domain 2 | Previously uncharacterized | Regulates miR-142 processing |
While the Molecular Chipper generates dense libraries for specific regions, other powerful CRISPR approaches have been developed for genome-wide studies of non-coding DNA.
A sophisticated method published in Nature Biomedical Engineering in 2024 enables systematic, large-scale deletion of non-coding regulatory elements (NCREs). This technique uses two sgRNAs working together to cut out both ends of a targeted NCRE, completely removing it from the genome6 .
In one massive screen, researchers used this system to target:
This approach identified numerous UCEs with essential biological functions, including one named PAX6_Tarzan, which was found to be critical for cardiomyocyte differentiation from human embryonic stem cells6 .
For studies where altering the DNA sequence is undesirable, scientists can use a catalytically inactive "dead" Cas9 (dCas9). This version still binds to DNA based on the guide RNA but does not cut it. By fusing dCas9 to effector domains, researchers can directly modulate gene activity2 :
This system is particularly valuable for studying how enhancers and silencers work in their native genomic context without permanently altering the DNA sequence2 .
| Research Tool | Function | Application in Non-Coding Studies |
|---|---|---|
| Cas9 Nuclease | Creates double-strand breaks in DNA | Disrupts regulatory elements via indels |
| dCas9-Fusion Proteins | Modulates transcription without cutting DNA | Tests enhancer/silencer function |
| Dual-sgRNA Vectors | Enables large deletions or genomic rearrangements | Removes entire regulatory elements |
| HDR Enhancers (e.g., L-755,507) | Improves precision editing efficiency | Inserts specific variants into regulatory regions |
| Next-Gen Sequencing | Detects editing outcomes and functional effects | Measures changes in chromatin accessibility and gene expression |
The implications of mapping the non-coding genome extend far beyond basic science. In 2025, researchers at the University of Oxford used precision CRISPR editing to identify a hidden switch within the TNFRSF1A gene, which encodes a receptor for tumor necrosis factor-alpha—a key player in autoimmune diseases like Crohn's disease and multiple sclerosis5 . By deleting a specific 3.3 kb region in human stem cells and differentiating them into macrophages, they demonstrated that this non-coding element functions as an enhancer that fine-tunes the receptor's expression5 .
Such discoveries illuminate why certain genetic variants predispose individuals to disease and may explain differences in patient responses to therapies. TNF inhibitors are widely prescribed biologics, and understanding how TNFRSF1A expression is regulated could inform why some patients respond better than others5 .
As CRISPR technologies continue to evolve—with innovations like Molecular Chipper for dense coverage, dual-CRISPR for systematic deletion, and single-cell multi-omics for linking genotype to phenotype—we are moving closer to a comprehensive functional map of the entire human genome4 6 8 . This map will not only transform our understanding of human biology but also pave the way for novel diagnostics and therapies for a vast range of genetic diseases.