A Major Step Forward with In Vivo CRISPR Screening Toward the Identification of Key Genes in Sperm Quality

Department of Molecular Cell Biology, University of California, Berkeley PostDoc
Program Specific Researcher with the Suzuki Lab, at the time of this research

Yuki Noguchi

Yuki Noguchi

Dr Noguchi is currently studying mechanisms of ovarian aging at the Lin He lab, University of California, Berkeley. Previously, as a PhD student in Professor Jun Suzuki’s lab at iCeMS, he developed an in vivo genome-wide screening method to identify genes that determine sperm quality in the mouse testis.

Please share with us the significance of your manuscript in terms of its research achievements, impact, or uniqueness.

A biological phenotype is the result of the complex interactions between gene expression and protein function within cells. Understanding these intricate molecular mechanisms is essential for gaining insights into physiological phenomena and diseases in the body. To achieve this, genome-wide screening methods have been developed to enable comprehensive analysis of approximately 20,000 genes at the same time, leading to the identification of various critical genes, including those involved in cancer progression. With the advent of the Nobel Prize-winning CRISPR-Cas9 system has further revolutionized these screenings, making them more accessible and easy.However, conventional genome-wide screening methods have typically been performed using cultured cells, making it difficult to apply them within the bodies of organisms like mice. In addition, since biochemical reactions in cell lines do not always align with those occurring in organs, performing screenings directly in mice would allow for a better understanding of molecular mechanisms relevant to physiological processes. Based on this idea, I established an in vivo genome-wide screening method using a CRISPR-sgRNA library, applying it to mouse testes to identify genes involved in spermatogenesis, and successfully identified Rd3 as a key factor in regulating sperm quality.

Please tell us what was the most gratifying or inspiring moment for you during this research project.

I still vividly remember the most exciting moment of this research—it was when I was analyzing the sequencing data from the screening results. After two years of meticulously overcoming each technical challenge, I sat in front of my computer, feeling a mix of excitement and nervousness. I was eager to see what kind of discoveries would emerge from the screening system I had painstakingly established, yet at the same time, anxious about the possibility of disappointing results. That blend of hope and uncertainty remains etched in my memory.

Please tell us about the biggest challenge or problem you experienced while conducting your research. How did you overcome it?

The greatest challenge in this study was understanding the biology of sperm and translating that knowledge into a workable experimental system. The screening method we developed consists of four key steps: (1) injecting a CRISPR-sgRNA library into the seminiferous tubules of mouse testes to efficiently induce gene knockouts in spermatogonial stem cells; (2) analyzing mature sperm using flow cytometry and sorting those with functional defects; (3) extracting genomic DNA from small number of sperm and identifying the incorporated sgRNA; and (4) creating a new sgRNA library based on the identified sgRNA and repeating steps 1-3.
For steps 1-3, there was little to no prior research to guide me. In step 1, I encapsulated the CRISPR-sgRNA in fine capillary glass and practiced injections into the seminiferous tubules of juvenile mice, often using resources like YouTube for technique. I also used Sendai virus vectors to efficiently introduce the library into spermatogonial stem cells. For step 2, I optimized the temperature, buffer, and other conditions to sensitively detect the biochemical signals of sperm, as there were no established protocols for flow cytometry in sperm analysis. Lastly, for step 3, I developed a technique to recover sgRNA from the densely packed genomic DNA of sperm, even when handling with very small sample sizes, without losing any sgRNAs.
Countless optimizations were necessary throughout the process, but by refining this experimental system, I gained a deeper understanding of sperm biology. In retrospect, this entire my challenge has been an invaluable learning experience.

Would you consider this work to be a turning point in your overall research direction? If so, how has your research direction changed as a result of this work?

Through this research, I learned the importance and enjoyment of carefully refining experimental conditions. As I mentioned in Question 3, the experience of optimizing the system by leveraging the unique characteristics of sperm has been invaluable. This approach and mindset are not only applicable to sperm but have also proven to be highly beneficial in my current work on ovarian aging and in building computational analysis systems. Additionally, this research sparked a deeper interest in reproductive biology and medicine, which I am now continuing to explore at the Lin He lab.

Please describe the current situation of your career. What is your current position? How has the knowledge and experience gained at iCeMS influencing your career?

I am currently a postdoc at the Lin He lab at the University of California, Berkeley. One of my most vivid memories from my time at iCeMS is that biology and chemistry labs coexisted on the same floor or in the same building. Although I haven’t experienced many research institutes, I found the interdisciplinary atmosphere at iCeMS to be inspiring and refreshing, and it constantly motivated me. Looking back, I do regret not chatting more with researchers from other fields. Additionally, the shared facilities were very well-equipped, which allowed me to conduct my research efficiently. I am deeply grateful to the staff for their support. Especially, I really appreciate for many support of the animal facility. Thank you so much!

Finally, please give a message or advice to young iCeMS researchers.

During my five years at iCeMS, the view of Hyakumanben from the window, which I saw almost every day, became such a great memory for me. I hope all of you also have a fulfilling and rewarding research experience at iCeMS.

Paper information

In vivo CRISPR screening directly targeting testicular cells

Yuki Noguchi, Yasuhito Onodera, Tatsuo Miyamoto, Masahiro Maruoka, Hidetaka Kosako, Jun Suzuki

Cell Genomics

Published: March 2024

DOI: 10.1016/j.xgen.2024.100510