Exploring the groundbreaking discovery of 13 testis-enriched genes that are surprisingly dispensable for reproduction
In the intricate dance of human reproduction, male fertility has long been a subject shrouded in biological mystery. For decades, the development of contraceptive options has largely focused on female biology, leaving a significant gap in reproductive healthcare.
of couples worldwide experience infertility
of infertility cases involve male factors
genes found dispensable for male fertility
The World Health Organization estimates that approximately 15% of couples worldwide experience infertility, with male factors being a primary or contributing cause in about half of these cases 7 . Despite this staggering statistic, our understanding of the genetic underpinnings of male reproduction remains surprisingly limited.
Enter CRISPR-Cas9, a revolutionary gene-editing technology that has transformed biological research. Often described as "molecular scissors," CRISPR allows scientists to make precise changes to DNA with unprecedented ease and accuracy.
This technology has opened new frontiers in our understanding of human genetics, including the complex biological processes required for male reproduction. In a fascinating twist, some of the most impactful scientific discoveries aren't about what genes do, but rather what they don't do—a paradox perfectly illustrated by a groundbreaking 2020 study that used CRISPR to identify 13 testis- or epididymis-enriched genes that are surprisingly dispensable for male fertility in mice 1 6 .
To appreciate the significance of these findings, we first need to understand the tool that made them possible. The CRISPR-Cas9 system is derived from a natural defense mechanism found in bacteria, which use it to protect themselves against viral invaders.
Acts as molecular scissors to cut DNA at precise locations determined by the guide RNA.
Directs the Cas9 scissors to a specific location in the genome through complementary base pairing.
The process is remarkably precise—the guide RNA, customized in the laboratory, carries a sequence that matches the gene researchers want to edit. When introduced into cells, this RNA seeker pairs with its corresponding DNA sequence, signaling the Cas9 scissors to make a clean cut at that exact location 3 8 .
Researchers identify the specific gene sequence they want to modify.
A custom RNA molecule is designed to match the target DNA sequence.
Guide RNA binds to Cas9 enzyme, forming the CRISPR-Cas9 complex.
The complex locates and cuts the target DNA sequence.
The cell's repair mechanisms introduce changes to the DNA sequence.
Once the DNA is cut, the cell's natural repair mechanisms kick in, allowing researchers to effectively disable or "knock out" specific genes 4 . This enables scientists to study what happens when a particular gene is missing—much like understanding a machine's function by removing one component at a time and observing the consequences.
What makes CRISPR particularly revolutionary is its simplicity, efficiency, and versatility. Unlike previous gene-editing technologies that were time-consuming, expensive, and required specialized expertise, CRISPR has democratized genetic research, accelerating the pace of discovery across countless fields, including reproductive biology 3 .
Armed with this powerful tool, researchers set out to investigate which genes are essential for male fertility. The research approach was both systematic and ingenious 1 :
Identified testis- or epididymis-enriched genes that are evolutionarily conserved between mice and humans.
Created genetically modified mice lacking each of the 13 target genes using CRISPR-Cas9.
Conducted comprehensive reproductive assessments to determine fertility impacts.
First, scientists identified genes that are "testis- or epididymis-enriched"—meaning these genes are predominantly active in the male reproductive tissues, specifically the testicles (where sperm is produced) and the epididymis (where sperm matures). This tissue specificity suggested these genes might play important roles in reproduction. Additionally, the team focused on genes that are evolutionarily conserved between mice and humans, indicating they likely serve fundamental biological functions that have been maintained through millions of years of evolution.
Collect newly fertilized mouse eggs
Deliver CRISPR components into zygotes
Transfer embryos to surrogate mothers
Assess genetic modifications and fertility
The experimental design was elegant in its simplicity: if you want to know what a gene does, see what happens when it's not there. Using CRISPR-Cas9, researchers created genetically modified mice lacking each of the 13 target genes. These "knockout" mice were then put through a series of comprehensive tests to assess their reproductive capabilities 1 .
The CRISPR components were introduced into mouse zygotes (newly fertilized eggs) through either microinjection or electroporation—techniques that help deliver genetic material into cells. These treated embryos were then implanted into surrogate mother mice, leading to the birth of founder generation animals carrying the desired genetic modifications 1 .
The findings challenged conventional wisdom in reproductive genetics. One might assume that genes highly active in reproductive tissues would be crucial for fertility, yet the research told a different story.
| Gene Name | Human Ortholog | Tissue Expression | Reproductive Phenotype |
|---|---|---|---|
| 4921507P07Rik | C7orf31 | Testis-enriched | Normal fertility |
| Allc | ALLC | Testis-enriched | Normal fertility |
| Cabs1 | CABS1 | Testis-enriched | Normal fertility |
| Fam229b | FAM229B | Testis-enriched | Normal fertility |
| Fscb | FSCB | Testis-enriched | Normal fertility |
| Iqca | IQCA | Testis-enriched | Normal fertility |
| Lelp1 | LELP1 | Testis-enriched | Normal fertility |
| Spata24 | SPATA24 | Testis-enriched | Normal fertility |
| Hdgfl1 | HDGFL1 | Testis-enriched | Normal fertility |
| Tmem97 | TMEM97 | Testis-enriched | Normal fertility |
| Lrcol1 | LRCOL1 | Epididymis-enriched | Normal fertility |
| Tmem114 | TMEM114 | Epididymis-enriched | Normal fertility |
| Eddm3b | EDDM3B | Epididymis-enriched | Normal fertility |
When the genetically modified male mice reached sexual maturity, researchers conducted detailed fertility tests. Each knockout male was paired with wild-type (genetically normal) females, and their reproductive performance was closely monitored for extended periods—typically 8 to 16 weeks 1 . The results were striking: all 13 knockout mouse lines exhibited normal fertility, producing similar numbers of offspring compared to their wild-type counterparts.
But the investigation went beyond simply counting babies. Researchers conducted comprehensive phenotypic analyses, examining testis appearance and weight, tissue histology, sperm movement and motility patterns, and sperm morphology. Across all these measurements, no significant abnormalities were detected in the knockout mice compared to their wild-type counterparts 1 .
Continuous mating with wild-type females showed normal litter size and frequency in all knockout mice.
Testis weight relative to body weight showed no significant difference between knockout and wild-type mice.
Histological staining of testis/epididymis sections revealed normal cellular organization.
Computer-assisted sperm analysis (CASA) showed normal motility parameters in knockout mice.
These genes, despite their specific expression in reproductive tissues and evolutionary conservation, were individually dispensable for male reproduction—a finding that has important implications for both basic science and contraceptive development.
Conducting such sophisticated genetic research requires an array of specialized tools and reagents. Here's a look at the essential components that made this fertility research possible:
| Tool/Reagent | Function | Application in Fertility Research |
|---|---|---|
| Cas9 Nuclease | DNA-cutting enzyme | Creates targeted breaks in gene sequences |
| Guide RNA (gRNA) | Targeting system | Directs Cas9 to specific genes of interest |
| Electroporation System | Delivery method | Introduces CRISPR components into zygotes |
| Microinjection Apparatus | Precision delivery | Injects CRISPR reagents into pronuclei of zygotes |
| Genomic Cleavage Detection Kit | Editing verification | Confirms gene knockout efficiency |
| Digital PCR | Expression analysis | Measures tissue-specific gene expression |
| Single-cell RNA-seq | Cellular mapping | Identifies gene expression in specific testicular cell types |
Uses electrical currents to create temporary pores in cell membranes through which CRISPR reagents can enter.
Involves manually injecting CRISPR components into fertilized eggs using extremely fine needles.
Once the genetically modified mice were created, various analytical tools came into play. Digital PCR and single-cell RNA sequencing helped verify tissue-specific gene expression patterns, while genomic cleavage detection kits confirmed that the target genes had been successfully disrupted. For fertility assessment, advanced systems like computer-assisted sperm analysis (CASA) provided precise measurements of sperm motility and function 1 4 .
In scientific research, we often celebrate discoveries of what matters, but understanding what doesn't matter is equally valuable. The finding that these 13 reproductively-enriched genes are dispensable for fertility represents what scientists call "negative results"—but these results are anything but unimportant.
Other genes may compensate for the loss of function (functional redundancy).
Helps identify poor targets for contraceptive development, saving resources.
Demonstrates the resilience of reproductive systems through evolutionary backup mechanisms.
From a basic science perspective, these findings help refine our understanding of genetic redundancy and compensation in biological systems. The fact that mice can reproduce normally without these genes suggests several intriguing possibilities: there might be other genes that can compensate for their loss (functional redundancy), or these genes might play more subtle roles that weren't detected under laboratory conditions, such as providing advantages in different environmental contexts 1 .
From a practical standpoint, these results help guide future research directions. For pharmaceutical companies interested in developing non-hormonal male contraceptives, these genes would be poor targets since disrupting them doesn't impair fertility. This knowledge prevents wasted resources and helps focus efforts on more promising candidates 1 6 .
This research also highlights the remarkable robustness of biological systems. Reproduction is such a fundamental process that evolution has built in numerous backup systems—when one genetic component is missing, others can often compensate to ensure the system continues to function.
Similar follow-up studies have continued to expand our understanding of reproductive genetics. A 2024 study identified 15 additional reproductive organ-enriched genes that are dispensable for male fertility 2 , and a 2025 paper added 18 more genes to this growing list 7 . Each of these "negative" results helps paint a more complete picture of the complex genetic landscape underlying male reproduction.
The application of CRISPR technology to study reproduction represents just the beginning of a new era in genetic research. As scientists continue to identify which genes are essential—and which are not—we move closer to understanding the core genetic requirements for fertility. This knowledge has far-reaching implications, from developing novel contraceptives to addressing male infertility, and even informing conservation efforts for endangered species.
What makes this research particularly exciting is its potential to translate from basic science to clinical applications. The same CRISPR tools used to understand reproductive genetics in mice are already being deployed in human clinical trials for various genetic disorders 5 . While much work remains before CRISPR-based reproductive treatments become commonplace, the foundational research being conducted today paves the way for tomorrow's medical breakthroughs.
In the end, the story of these 13 "dispensable" genes reminds us that in science, sometimes what we don't find is as important as what we do. As CRISPR technology continues to evolve, it will undoubtedly uncover more of reproduction's genetic mysteries—both the essential players and the surprising bystanders—bringing us closer to a comprehensive understanding of one of life's most fundamental processes.