Biological and Medicinal Chemistry

CRISPR-Click Enables Multi-Gene Editing with Modular Synthetic sgRNAs

Authors

Abstract

Gene editing systems such as CRISPR/Cas9 readily enable individual gene phenotypes to be studied through loss-of-function. However, in certain instances, gene compensation can obfuscate the results of these studies, necessitating the editing of multiple genes to properly identify biological pathways and protein function. Performing multiple genetic modifications in cells remains difficult due to the requirement for multiple rounds of gene editing. While fluorescently labeled guide RNAs (gRNAs) are routinely used in laboratories for targeting CRISPR/Cas9 to disrupt individual loci, technical limitations in single guide RNA (sgRNA) synthesis hinder the expansion of this approach to multi-color cell sorting. Here, we describe a modular strategy for synthesizing sgRNAs where each target sequence is conjugated to a unique fluorescent label, which enables fluorescence-assisted cell sorting (FACS) to isolate cells that incorporate the desired combination of gene-editing constructs. We demonstrate that three short strands of RNA functionalized with strategically placed 3’-azide and 5’-alkyne terminal deoxyribonucleotides can be assembled in a one-step, template-assisted, copper-catalyzed alkyne-azide cycloaddition (CuAAC) to generate fully functional, fluorophore-modified sgRNAs. Using these synthetic sgRNA in combination with FACS, we achieved selective cleavage of two targeted genes, either separately as a single color experiment or in combination as a dual-color experiment. These data indicate that our strategy for generating doubly-clicked sgRNA allows for Cas9 activity in cells. By minimizing the size of each RNA fragment to 41 nucleotides or less, this strategy is well suited for custom, scalable synthesis of sgRNAs.

Content

Thumbnail image of Park_Manuscript.pdf

Supplementary material

Thumbnail image of Park_supporting information.pdf
Supporting information of CRISPR-Click Enables Multi-Gene Editing with Modular Synthetic sgRNAs
Supporting information of "CRISPR-Click Enables Multi-Gene Editing with Modular Synthetic sgRNAs"