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

CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, the Cas9 endonuclease is guided towards its DNA target sequence by the guide RNA (gRNA). Chemical synthesis of a functional 97-nucleotide single gRNA (sgRNA) is non-trivial 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 RNases. Modified nucleotides consisted of a 2O-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 knock-out 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 disrupting gene expression of two proteins.