Jun 25, 2024

In the human genome, only 2% of the DNA sequences code for proteins, while the vast majority of the sequences have no coding function. Whether these non-coding sequences have actual functions has been a major focus of biological research in recent years.

With the development of life science technology, scientists have gradually discovered that some non-coding sequences play an important role in regulating the expression of downstream genes. These sequences are called non-coding regulatory elements (NCREs). Unfortunately, the scientific community still lacks a powerful tool to systematically study NCREs. However, with the advent of the CRISPR-Cas9 system, rapid and precise gene editing has become possible.

On May 22, 2024, researchers from Leiden University in the Netherlands published a paper in the Nature Biomedical Engineering journal titled "Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs." In the paper, they reported on a method they developed using the Cas9 system to efficiently study the biological functions of NCREs.

Dual-CRISPR System

To achieve efficient gene editing and screening, the researchers introduced two gRNAs into the CRISPR system, targeting the 5' and 3' ends of the target sequence, respectively, and integrated them into the same plasmid in an opposite direction. The advantage of this dual-CRISPR system is that it makes high-throughput sequencing more convenient after transfection and can quickly screen out invalid lentiviral systems. These advantages make it possible to high-throughput edit tens of thousands of NCREs.

Even with a good high-throughput editing tool, researchers cannot use an exhaustive method to study the vast non-coding sequences. Therefore, the researchers initially selected 4047 highly conserved elements (Ultra-Conserved Elements, UCEs) and 1527 enhancers that have been validated in vitro, and designed over 60,000 dual-CRISPR sequences. By editing the K562 system and analyzing the whole-genome knockout algorithm, the researchers screened out 346 UCEs that may play a key role in K562 cell growth. UCEs and NCREs (de_novo_1) such as PBX3_Cl, FOXP1_Fl, PAX6_Ta showed significantly slower cell growth after being knocked out in cells. To the researchers' surprise, several elements did not exhibit enhancer function in the luciferase system, and PBX3_Cl and de_novo_1 also exhibited silencer activity.

To explain the strange phenomenon in the luciferase system, the researchers analyzed the genes within the topological associated domain (TAD) of these elements. They found that the PTPRD and RCN1 genes within the TAD were significantly upregulated after PAX6_Ta and de_novo_1 were knocked out. Regulating the PTPRD and RCN1 genes could reproduce the phenotype of knocking out PAX6_Ta and de_novo_1, proving that these UCEs may affect cell growth through PTPRD and RCN1.

Using similar research methods, the researchers explored the origin of the TKI inhibitor imatinib resistance in leukemia and found that cells knocked out of ZNF503_Op and QKI_Jo exhibited significant drug resistance. Further analysis revealed that ZNF503_Op and QKI_Jo could act as silencers of SAMD8, ZNF503 and PACRG, allowing imatinib to function normally. Clinical mutations in the QKI_Jo region can significantly reduce the sensitivity of patients to imatinib, and these data further demonstrate the application prospects of the dual-CRISPR tool.

Taking It to the Next Level

Since the researchers wanted to design 250,000 gRNAs to explore the function of over 13,000 potential enhancers, the original screening efficiency was a bit stretched. Therefore, the research team optimized their tools. The efficiency of NGS was further improved by increasing the distance between the two gRNAs.

With the iterative tool, the research team found 1005 enhancers that could affect cell growth and studied 6 of the highest-scoring potential enhancers. Experimental results showed that the function of these six elements varied greatly depending on the promoter. At the same time, these enhancers may affect cell growth by influencing three genes, ZNF263, PATZ1 and KLF4. And the enhancer E22:23590 located in the BCR region may affect cell growth by influencing the BCR-ABL fusion gene and Rab36. These results further demonstrate the powerful function of dual-CRISPR-2.0.

Single-Cell Application

Despite screening a large number of enhancers, only a few were found to affect cell growth. This led the researchers to hypothesize that many enhancers are "redundant" and only function when multiple enhancers work together to influence cell growth. To investigate this further, they grouped the enhancers and studied the three groups with the highest scores in detail.

Using an enhancer group on chromosome 22 as an example, they found that knocking out any single enhancer did not affect cell growth. However, knocking out any two or all three enhancers significantly reduced cell growth. This demonstrates that dual-CRISPR-2.0 can be used to study the interactions between multiple NCREs.

To extend their analysis, the researchers applied the tool at the single-cell level. The presence of dual gRNAs facilitated the identification of edited cells, allowing them to observe similar results as before. Notably, they saw the same impact of PAX6_Ta knockout on genes within its TAD. Furthermore, they identified single-cell-specific enhancers and their corresponding regulatory genes, validating the technique's single-cell functionality.

Clinical Relevance of PAX6_Ta

After repeatedly demonstrating the effectiveness of dual-CRISPR-2.0 in genome-wide NCRE research, the researchers selected the PAX6_Ta element, which had been identified multiple times, for further investigation to establish the method's clinical relevance. Knocking out PAX6_Ta in human embryonic stem cells (hESCs) significantly impaired their ability to differentiate into cardiomyocytes. In the small group of cells that successfully differentiated into cardiomyocytes, PAX6_Ta knockout significantly disrupted their beating behavior. Correspondingly, the expression of RCN1 and PAX6 was significantly upregulated. These genes have previously been reported to play crucial roles in cardiomyocyte formation and function. These findings suggest that PAX6_Ta, as an NCRE, may play an important role in cardiomyocyte differentiation, providing a new direction for the development of heart-related therapies.

This study employed a novel dual-CRISPR system to achieve high-throughput analysis of non-coding regulatory elements in the genome and successfully identified numerous NCREs with previously unknown functions. This provides a powerful tool for future NCRE research.

Reference

[1] Li, Y., Tan, M., Akkari-Henić, A. et al. Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs. Nat. Biomed. Eng (2024). https://doi.org/10.1038/s41551-024-01204-8