Use of Synthetic sgRNAs for Improved CRISPR Editing in Various Cell Types [POSTER]

This poster examines the following:

  • Value of RNP Formation
  • More Consistent Guides with Synthetic RNA
  • Two-Piece vs. One-Piece Systems
  • Functional Validation Across Cell Types
  • Importance and Potential Benefits of Chemical Modification

Full Abstract
CRISPR/Cas9 technology has made genome editing accessible for a wide range of cell-types and model organisms. However, obtaining consistent editing efficiencies remains a challenge. Recent studies have shown that forming a ribonucleoprotein (RNP) complex between Cas9 nuclease and guide RNAs produces the most consistent genome edits with the lowest off-target effects and no risk of DNA incorporation compared to plasmid or mRNA-based approaches.

When forming RNPs, the quality of the guide RNA is critical for achieving consistent editing efficiencies. Traditional methods for generating guide RNAs, using in vitro transcription (IVT), yields sgRNA molecules of inconsistent length and quality, which can affect genome editing efficiency. The use of chemically synthesized guide RNAs provides a more consistent option for generating RNPs, and is more amendable to scale-up.

Until now, synthetic RNA for CRISPR was only available as a two-part system that requires pre-annealing and is prone to inconsistencies with annealing and thus editing efficiencies. Synthego has developed novel RNA synthesis hardware that is capable of producing the world’s first high quality 100-mer single guide RNA (sgRNA) for CRISPR at a practical scale and price. Here we demonstrate that Synthego sgRNA can produce consistent genome editing efficiencies that are superior to two-piece crRNA:tracrRNA complexes and act as a drop in replacement for IVT-derived guides, while providing superior scalability and minimal labor cost. Functional validation has been achieved in a variety of cell types and systems including: adherent mammalian cell lines, primary T-cells, iPSCs and model organism embryos.

Furthermore, chemical modification of 5’ and 3’ terminal sgRNA residues with 2’-O-methyl and 3’ phosphorothioate internucleotide linkages are shown to provide additional improvements in editing efficiency for particular cell types and genomic targets that can prove otherwise challenging to edit.