Part:BBa_K5310030

INTRODUCTION

The advent of CRISPR-Cas9 technology has revolutionized gene editing, allowing for precise modifications in the genome. Specifically, the use of single-guide RNAs (sgRNAs) enables targeted gene activation or disruption, offering a promising approach to regulate gene expression and address autoimmune conditions such as multiple sclerosis (MS). In our project “NeuroMuSceteer”, we aim to leverage sgRNAs to epigenetically induce FOXP3 activation, transforming cytotoxic T cells into regulatory T cells (T-regs) that enhance immune safety and functionality.

MECHANISM OVERVIEW

The mechanism involves the design and application of specific sgRNAs that target the FOXP3 gene. Each sgRNA is carefully constructed to ensure high specificity and efficiency in guiding the Cas9 nuclease to the desired genomic locus. By employing the CRISPick tool, we selected unique epitopes within the FOXP3 gene, from which we developed complementary sequences. This step is critical as it lays the groundwork for successful gene modification. Additionally, each sgRNA is configured in the correct orientation (5' to 3') to facilitate optimal binding and activity of the CRISPR machinery.

To allow for efficient cloning and insertion into the plasmid backbone, we incorporated BsmBI restriction enzyme sequences at both ends of the sgRNAs. This inclusion not only enables precise integration into the plasmid but also ensures that the sgRNAs can be effectively expressed in K562 cells. The overall design guarantees that the CRISPR-Cas9 system will successfully activate FOXP3 expression, promoting the desired transformation of T cells and enhancing their regulatory functions.

PART FUNCTION

This sgRNA part "Oligo 4" (BBa_K5310030) is designed to play a pivotal role in our transformation strategy by facilitating the targeted modification of the FOXP3 gene. It is complementary to "Oligo 3" (BBa_K5310029), allowing for precise binding and effective cleavage by the Cas9 nuclease.

REFERENCES

Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014 Aug;11(8):783-784. doi: 10.1038/nmeth.3047.