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

Disrupting Protein Expression with Double-Clicked sgRNA–Cas9 Complexes: A Modular Approach to CRISPR Gene Editing. Santiago Tijaro-Bulla, Eiman A. Osman, Chris D. St Laurent, Kelli A. McCord, Matthew S. Macauley, and Julianne M. Gibbs. ACS Chemical Biology 2023 18 (10), 2156-2162DOI: 10.1021/acschembio.3c00140

    Abstract

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

    Territorial Acknowledgement

    The University of Alberta, its buildings, labs, and research stations are primarily located on the traditional territory of Cree, Blackfoot, Métis, Nakota Sioux, Iroquois, Dene, and Ojibway/Saulteaux/Anishinaabe nations; lands that are now known as part of Treaties 6, 7, and 8 and homeland of the Métis. The University of Alberta respects the sovereignty, lands, histories, languages, knowledge systems, and cultures of First Nations, Métis and Inuit nations.

    Scroll to Top