Summary of Prime Editing (PE)

PE is an accurate glycogenomics editing technology based on the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system and it does not need exogenous donor DNA and DNA double-strand breaks (DSBs). PE depends on the "search" and "replacement" of target sequences. nCas9 with single-strand cleavage activity was blended into M-MLV-RT and targeted to a specific site mediated by prime editing guide RNA (pegRNA) in the PE system. Then, the single strand at the target site is cleaved and nicked by the effector protein, and reverse transcription begins dependent on the reverse transcription template (RTT) in the pegRNA. Next, the edited DNA sequence is integrated into the complementary strand by the DNA repair mechanism, and the edited DNA is further copied into the other strand, resulting in a stable edited DNA sequence.

Fig.1 Schematic diagram of the operating principle based on the prime editing system. (Huang & Liu, 2023)

PE-based Glycogene Editing Technology at CD BioGlyco

PE is the most practical of the editing tools due to its high efficiency in a two-plasmid system comprising a nickase Cas9-RT fusion and a pegRNA plasmid.

• Glycogene Editing Service
  
  • Targets genome deletion
  • Replacement
  • Insertion of the targets
• Delivery Service of PE Agents
  In addition to editing efficiency, successful genome editing also depends on the efficiency of delivery to the target.
  
  • Virus-based delivery: A series of viral vectors have been used to deliver genome editing tools, including adeno-associated viral (AAV), lentiviral, retroviral, adenoviral, and baculoviral vectors.
  • Non-viral delivery: It is suitable for transient expression of genome editing tools and has low DNA toxicity. Most importantly, non-viral platforms hold promise for packaging PE formulations as they can be delivered without packaging volume constraints. Cas9 ribonucleoprotein (RNP) is a popular non-viral platform that has been directly used to deliver PE.
  • Algorism development for the design of pegRNA: In exploring the diversity of PE applicability across species, it is more convenient to incorporate tools for PE system design and editing effect prediction.

Fig.2 PE experiment procedure. (CD BioGlyco)

Applications

• In plants: Provide better technical support for crop breeding improvement.
• In animals: The non-mammalian and mammalian animal model insect.
• In microorganism: Build the model of the microorganism E. coli; PE has shown great potential in engineering cell lines or strains with new functions or phenotypes and creating new platforms that are used for producing enzymes, chemicals, and so on.

Advantages

• Fewer constrained by protospacer adjacent motif (PAM) sequence location
• Reduce the off-target editing, less indel by-products, and more versatile and precise
• Do not need DSB or exogenous donor DNA
• High editing accuracy, high efficiency, and flexibility
• Increased the scope of genome editing.
• Extraordinary versatility, product purity, and target specificity.

CD BioGlyco has been committed to Glycogenomics research for many years and has accumulated rich practical experience. Please feel free to contact us for more information about genetic research.

References:

1. Huang, Z.; Liu, G. Current advancement in the application of prime editing. Front Bioeng Biotechnol. 2023, 16(11): 1039315.
2. Lu, C.; et al. Prime editing: an all-rounder for genome editing. Int J Mol Sci. 2022, 23(17): 9862.