Disrupting Protein Expression with Double-Clicked sgRNA–Cas9 Complexes: A Modular Approach to CRISPR Gene Editing

• DOI: 10.1021/acschembio.3c00140
• Publication Date: 2023-08-09T17:15:14Z
• Authors: Santiago Tijaro-B...; Eiman A. Osman; Chris D. St Laurent; Kelli A. McCord; Matthew S. Macauley; Julianne M. Gibbs
• Keywords: Guide RNA; Subgenomic mRNA

CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, Cas9 endonuclease guided toward its DNA target sequence by guide RNA (gRNA). Chemical synthesis of a functional single gRNA (sgRNA) nontrivial because length strand. Recently we demonstrated that sgRNA can be stitched together from three smaller fragments through copper-catalyzed azide-alkyne cycloaddition, making process highly modular. Here further advance approach leveraging modulator platform incorporating chemically modified nucleotides at both ends modular increase resistance against ribonucleases. Modified consisted 2'-O-Me group and phosphorothioate backbone varying number 5'- 3'-ends sgRNA. It was observed significantly increased success knocking out interest. Using these stabilized sgRNAs facilitates multigene protein level, as successful knockout Siglec-3 Siglec-7 using two fluorophores conjunction with fluorescence-activated cell sorting. These results demonstrate versatility for assembling small, strands simultaneously disrupt expression proteins.