Difference between revisions of "Part:BBa K4830007"

Revision as of 17:09, 10 October 2023

HEK3 ngRNA

HEK3 ngRNA - nicking guide RNA targeting the gene encoding EPHA8 (EPH Receptor A8)

Usage and Biology

Prime editing is an innovative technology for genome editing that enables the installation of the wide spectrum of gene modifications such as 12 possible base-to-base conversions, small insertions, and deletions, without requiring double-stranded breaks or donor DNA templates. This technology provides high versatility and target specificity, offering the potential to revolutionize medicine by providing novel tools for treating genetic diseases.

Prime editing relies on specialized prime editors, which usually consist of reverse transcriptase enzyme fused to nickase Cas9, and prime editing guide RNA containing a spacer that specifies the target site. It also includes a scaffold and 3’ extension containing a primer binding site (PBS) and an RT template encoding the desired edit. To initiate prime editing, PE creates a single-strand break in the DNA at the target site to allow reverse transcriptase to access the DNA and synthesize a new DNA strand using pegRNA as a template. Then the information from the edited strand is copied to the complementary strand through the cell’s natural repair pathways. PE3b has an additional single guide (sgRNA) that nicks the unedited strand at a location away from the editing site. PE3b sgRNAs are designed with spacers to match the edited strand but not the original allele. By doing this, mismatches between the spacer and the unedited allele should disfavor sgRNA nicking until after editing of the PAM strand has occurred.

EPHA8 (EPH Receptor A8) is a gene encoding a member of the ephrin receptor subfamily of the protein-tyrosine kinase family. EPH and EPH-related receptors participate in development of the mammalian nervous system.

Characterization

The ngRNA in combination with pegRNA were used to test the efficiency of the Prime Editor containing the alternative Reverse Trancriptases. The pegRNA served as template to install the edit, in this case CTT nucleoide sequence insertion at the HEK3 target site. 3 plasmids containing PE, pegRNA and ngRNA were trasfected into HEK293T cells, and editing efficiency was evaluated 72hr after transfection using Sanger sequencing.

Insert graphs.

Figure 1 shows the editing efficiency of the alternative RTs on HEK3 target site, proving the validity of the pegRNA design.

Sequence and Features