Genome Engineering Using Multiple CRISPR-Cas Systems

The study, "Orthogonal Transcriptional Modulation and Gene Editing Using Multiple CRISPR-Cas Systems" explores the application of multiple RNA-delivered CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) effectors to achieve orthogonal modulation of gene expression.

By systematically screening a variety of transcriptional activators and repressors, the researchers identified dSpCas9-VPR as the most potent transcriptional activator and observed significant gene-specific variability, which was influenced by epigenetic factors.

The study also evaluated the repression efficiencies of different CRISPRi effectors, highlighting distinct performance differences, particularly in dSaCas9-based systems. Furthermore, the integration of CRISPRa, CRISPRi, and gene knockout into a trimodal engineering system was demonstrated, showcasing its potential for applications in primary human T cells.

The research provides critical insights into off-target effects, kinetic considerations, and the therapeutic and synthetic biology applications of these systems. These findings underscore the potential of multiplexed CRISPR systems for precise and versatile genetic engineering in diverse biological contexts.

CRISPR-Cas systems have revolutionized the fields of gene editing and transcriptional regulation, enabling precise and programmable genetic manipulations. This study focuses on the implementation of orthogonal CRISPRa and CRISPRi using multiple Cas9 orthologs, which allows for simultaneous transcriptional activation, repression, and gene knockout within the same cell.?

The ability to independently control these processes is essential for optimizing genetic interventions in complex biological systems. Applications of such orthogonal systems span a wide range of fields, including immunotherapy, regenerative medicine, and synthetic biology. Understanding the interplay between these systems, as well as their kinetic and epigenetic influences, is critical for advancing their utility in both research and therapeutic settings.

To investigate the efficiency of RNA-delivered CRISPRa and CRISPRi effectors in modulating gene expression, the researchers employed a comprehensive screening approach. The methodology included the following steps:

1. Comparison of CRISPRa Systems: Direct Cas9 fusions and SunTag systems were compared to evaluate their transcriptional activation efficiencies.
2. Evaluation of CRISPRi Effectors: Different repressors were tested with dSpCas9 and dSaCas9 to assess their repression efficiencies and identify optimal configurations.
3. Orthogonal Delivery Methods: Strategies to prevent sgRNA cross-complexation were investigated to ensure the specificity and independence of multiplexed CRISPR systems.
4. Off-Target Effects and Specificity Analysis: High-throughput sequencing and transcriptomic analyses were performed to assess off-target effects and confirm the specificity of CRISPRa and CRISPRi.
5. Trimodal Engineering System: A system integrating CRISPRa, CRISPRi, and gene knockout was developed and tested to evaluate its feasibility and efficiency.
6. Cell-Based Testing: The systems were tested in both Jurkat cells (a model T cell line) and primary human T cells to assess their applicability across different cell types and their potential for therapeutic use.

The study yielded several key findings:

1. CRISPRa Efficacy: dSpCas9-VPR emerged as the most potent transcriptional activator. Gene-specific variability was observed, with the direct Cas9 fusion system outperforming the SunTag system for certain targets, such as CD19. Epigenetic status was found to significantly influence CRISPRa efficiency, with some genes exhibiting resistance to activation due to closed chromatin states.
2. CRISPRi Efficacy: Repression efficiencies varied across different CRISPRi effectors. While dSpCas9-based systems showed consistent repression, dSaCas9-KOX1 demonstrated superior repression of CD3e, likely due to differences in binding kinetics and chromatin accessibility.
3. Delivery Modalities: The potency of CRISPRi effectors was influenced by delivery methods, highlighting the importance of optimizing reagent delivery for specific applications.
4. Orthogonal Systems: The use of orthogonal CRISPRa and CRISPRi systems successfully prevented sgRNA cross-complexation, enabling independent modulation of multiple targets.
5. Multiplexed Systems: Multiplexed CRISPRa and CRISPRi exhibited target-specific and modality-dependent kinetics. However, CD3e repression efficiency was reduced in multiplexed settings, suggesting gene-specific susceptibility to reagent limitations.
6. Off-Target Effects: High specificity was confirmed for CD123 CRISPRa and CD5 CRISPRi, with no global transcriptomic alterations detected. Differentially expressed genes were attributed to downstream signaling effects rather than direct off-target activity.
7. Trimodal Engineering: The integration of CRISPRa, CRISPRi, and gene knockout using truncated sgRNAs enabled SpCas9 to perform multiple functions without nuclease activity at CRISPRa loci. While CRISPRa efficiency was slightly reduced with truncated sgRNAs, the system demonstrated robust performance in both Jurkat cells and primary human T cells.
8. Primary T Cell Applications: The trimodal system showed comparable efficiency in primary human T cells, with no significant impact on cell viability, proliferation, or cytokine secretion, except for a reduction in TNF-alpha secretion. These findings highlight the importance of optimizing sgRNA design and delivery for therapeutic applications.

The research confirms the feasibility of orthogonal transcriptional modulation using multiple CRISPR-Cas systems and provides several key insights:

1. Optimization of sgRNA and Delivery: The selection of sgRNAs and delivery conditions must be tailored to specific cell types and target genes to maximize efficiency and minimize off-target effects.
2. Kinetic Considerations: The temporal dynamics of CRISPRa and CRISPRi differ, emphasizing the need for precise temporal regulation in multiplexed systems.
3. Truncated sgRNAs: The use of truncated sgRNAs offers a promising approach for fine-tuning CRISPRa efficiency and kinetics, particularly in trimodal systems.
4. Balancing Components: Achieving an optimal balance between CRISPR components is critical to avoid reduced efficacy due to unsaturated Cas9 enzymes or excess sgRNA.
5. Epigenetic Influences: The epigenetic landscape of target genes plays a significant role in determining the efficiency of CRISPRa and CRISPRi, underscoring the need for epigenetic profiling in experimental design.

This research demonstrates the versatility and specificity of orthogonal CRISPRa and CRISPRi systems in gene regulation. The integration of trimodal engineering, combining CRISPRa, CRISPRi, and gene knockout, offers a powerful approach for advanced genetic manipulations. These systems hold significant promise for applications in immunotherapy, regenerative medicine, and synthetic biology. Future research should focus on optimizing delivery conditions, expanding the repertoire of Cas9 orthologs, and investigating the epigenetic influences on CRISPR-mediated transcriptional regulation. By addressing these challenges, the potential of multiplexed CRISPR systems for precise and scalable genetic engineering can be fully realized.

Image Credit: MicroOne/Shutterstock.com

Broks? AD, Bendixen L, Fammé S, Mikkelsen K, Jensen TI, Bak RO. Orthogonal transcriptional modulation and gene editing using multiple CRISPR-Cas systems. Mol Ther. 2025 Jan 8;33(1):71-89. doi: 10.1016/j.ymthe.2024.11.024. Epub 2024 Nov 19. PMID: 39563029; PMCID: PMC11764084.