Type I CRISPR-Cascade: precise gene modulation platform for human cell applications

Unmet Need

The gene editing sector has experienced a transformative surge in technological advancements with the emergence of CRISPR-Cas systems. These systems offer unprecedented capability to modify and modulate genetic sequences, proving instrumental from fundamental science to medical applications. Class 2 CRISPR-Cas systems, utilizing single-effector proteins such as Cas9 and Cas12a, have simplified genome editing by using RNA-guided nucleases that eliminate the need for complex protein engineering. However, despite the scientific community’s widespread adoption of CRISPR-Cas9 for its versatility, this and other Class 2 systems have limitations, including restrictive protospacer adjacent motifs (PAM) requirements and off-target activities. These limitations lead to undesirable outcomes such as reduced targeting flexibility and increased risk of unintended genetic alternations. In contrast, Class 1 CRISPR-Case systems, with their multi-subunit effector composition, form a complex known as Cascade. Despite representing approximately 90% of CRISPR-Cas systems, their complexity has hindered widespread adoption by scientists. However, this complexity allows for a more refined interaction with genetic material. This enables potentially broader gene manipulation capabilities and offers promising solutions to address the Class 2 limitations. There is a need for a more flexible and precise Class 1 CRISPR gene modulation system that offers a broader targeting PAM range, higher specificity in target recognition, and reduced off-target interactions.

Technology

Duke inventors have developed a gene modulation system for the precise control of targeted gene expression in human cells. This is intended for use in biomedical and biotechnological research, as well as in therapeutic R&D. Specifically, the innovation includes Type I variants of Class 1 CRISPR-Cas systems repurposed for targeted gene regulation in human cells. The system comprises multi-component RNA-guided complexes, known as Cascade, from E. coli and L. monocytogenes. These complexes have been shown to target specific loci of the human genome with programmable CRISPR RNA (crRNA).These complexes also achieve precise gene modulation through fusion with either activation or repression domains. Hence, the developed system can activate, repress, or edit desired gene functions and integrate seamlessly into existing research, product development, and clinical application workflows. This has been demonstrated to be effective in vitro in human cells, offering diverse PAM recognition sequences compared to traditional Class 2 CRISPR systems. Its multi-subunit configurations enhance targeting specificity and reduce off-target effects, showcasing its advanced precision.

Other Applications

This technology could also be utilized in diverse areas such as streamlining gene regulatory network design, illuminating distant genetic controls, enhancing gene silencing, and advancing targeted gene therapies.Bottom of Form

Advantages

• Novel type of CRISPR-based technology expands the toolbox for engineering human genomes
• Broader PAM sequence recognition expands the range of genetic targets
• Enhanced targeting specificity reduces off-target effects
• Modulation capability for both gene activation and repression applications