New CRISPR Tool Reveals Key Lymphoma Genes

By Dr. Chinta SidharthanReviewed by Lily Ramsey, LLMFeb 7 2025

Genetic engineering has revolutionized biomedical research, but optimizing these gene-editing tools remains a challenge. Scientists have now developed a novel approach using an enhanced version of the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12a (Cas12a) system in mice, enabling large-scale genetic screening with high efficiency.

A recent study published in Nature Communications explored how the CRISPR-Cas12a system, combined with newly designed compact CRISPR libraries, can identify genes crucial to cancer development. By investigating gene interactions in lymphoma models, the researchers also uncovered potential therapeutic targets.

Study: Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening. Image Credit: ArtemisDiana/Shutterstock.com

CRISPSR-Cas Systems

CRISPR-based genetic screening has become a vital tool for identifying genes involved in diseases, particularly in cancer research. Traditionally, CRISPR-Cas9 has been the predominant system for gene editing, but limitations such as sequence constraints and off-target effects have driven the search for alternative tools.

CRISPR-Cas12a, a newer enzyme, offers numerous advantages, including smaller guide ribonucleic acids (RNAs) and improved specificity.

However, its application in whole-genome screening remains underdeveloped. Scientists have worked on improving CRISPR-Cas12a’s efficiency to make it a more viable tool for studying complex genetic interactions.

Understanding gene networks in cancer, such as lymphoma, requires scalable methods capable of targeting multiple genes simultaneously. While previous research has used CRISPR-based knockout screens to identify cancer-driving mutations, gaps remain in optimizing this technology for in vivo applications.

About the Study

To develop a more efficient CRISPR screening system, the research team generated a transgenic mouse model expressing an enhanced Acidaminococcus-derived Cas12a called enAsCas12a. This system allowed for sustained gene-editing activity across different tissues.

The researchers further validated enAsCas12a’s efficacy through in vitro experiments using mouse fibroblast and lymphoma cell lines, as well as in vivo experiments using fetal liver cell transplants.

Two novel CRISPR-Cas12a libraries, named Menuetto and Scherzo, were created to perform large-scale genetic knockout screens. The Menuetto library was designed to enhance target coverage, while the Scherzo library was optimized for screens with limited cell numbers.

These libraries were introduced into lymphoma-prone mice to identify genes that influence cancer progression.

To conduct the experiments, the researchers transduced fetal liver cells from genetically engineered mice with non-targeting control guides, guides that targeted the transformation-related protein 53 (Trp53) gene or the Menuetto library. The transduced cells were then transplanted into irradiated wild-type recipient mice.

Tumor development was then monitored over time, and the researchers used deoxyribonucleic acid (DNA) sequencing to identify enriched pre-CRISPR RNAs (crRNAs) from lymphoma tissues to determine the genes essential to lymphoma progression.

Additionally, the study also involved flow cytometry analysis of blood, spleen, and bone marrow samples to characterize immune cell populations.

The researchers also used analyses such as Western blotting to assess gene disruptions in tumors, and in vitro screening was conducted in lymphoma cells treated with chemotherapy-like agents to determine gene function in drug response.

Major Findings

The results revealed that the newly developed enAsCas12a system successfully supported robust gene editing across different cell types, including blood and lymphoma cells. Furthermore, the use of the Menuetto and Scherzo CRISPR libraries enabled genome-wide knockout screening with high specificity and efficiency.

One of the key findings was the identification of Trp53 as a major driver of lymphoma progression. Mice with pre-crRNAs that targeted the Trp53 gene developed aggressive tumors significantly faster than controls, confirming the gene’s critical tumor-suppressor role.

Additional genes linked to lymphoma, such as transcription factor AP-4 (Tfap4), interferon regulatory factor 4 (Irf4), cyclin-dependent kinase inhibitor 2A (Cdkn2a), and solute carrier family 23 member 2 (Slc23a2), were also enriched in tumor samples, highlighting their potential involvement in cancer development.

Furthermore, the in vitro drug sensitivity tests further validated the screen’s findings, revealing that genes such as B-cell lymphoma 2-associated X protein (Bax) and p53 upregulated modulator of apoptosis (Puma) contribute to chemotherapy-induced apoptosis in lymphoma cells.

The study also demonstrated that the Menuetto library’s dual-guide system provides superior statistical robustness compared to traditional CRISPR screening methods.

Despite these promising results, there were a few limitations to this study. The research was focused on a murine model, and differences in gene function between mice and humans may affect the translatability of the findings.

Additionally, the study’s design prioritized loss-of-function mutations, potentially overlooking genes whose activation may also contribute to disease. However, the enAsCas12a system developed in this study has provided an efficient genetic screening tool for future in vitro and in vivo studies on cancer and other diseases.

Conclusions

Overall, the findings advanced existing CRISPR screening technology by integrating an optimized Cas12a system with compact, efficient libraries for genetic analysis.

Furthermore, by identifying key genes involved in lymphoma progression, the study highlighted new potential targets for cancer therapy.

These findings indicated that enAsCas12a is a powerful tool for studying gene function and disease mechanisms, with promising implications for future biomedical research and therapeutic development.