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Applications of chemically modified synthetic guide RNA for CRISPR-Cas9 genome editing

Applications of chemically modified synthetic guide RNA for CRISPR-Cas9 genome editing content piece image

The bacterial CRISPR-Cas9 system has been applied in mammalian cells to efficiently disrupt genes through the formation of targeted DNA double-strand breaks. The Cas9 nuclease is directed to DNA using a guide RNA (gRNA), either as the native dual-RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA), or a chimeric single guide RNA (sgRNA) created through the fusion of crRNA and tracrRNA. DNA-free genome engineering can be achieved by using Cas9 mRNA or Cas9 protein with a gRNA, including in vitro transcribed (IVT) gRNA, synthetic sgRNA or synthetic crRNA:tracrRNA. While IVT sgRNAs can elicit an immune response, synthetic sgRNA or crRNA:tracrRNA have little to no effect on the immune response and permit chemical modifications to be incorporated to the RNA for increased stability. Here we present chemical modification of synthetic crRNA:tracrRNA with one to three 2’-O-methyl and phosphorothioates (MS) on the 5’ and/or 3’ ends. These modified RNAs were co-delivered into cells with Cas9 mRNA or Cas9 protein using electroporation. Some modification patterns were found to significantly improve CRISPR-Cas9 gene editing when used with Cas9 mRNA compared to the unmodified versions, yet most modifications did not significantly increase gene editing when used with Cas9 protein. Transfection reagent-mediated delivery of these modified gRNAs into a Cas9-expressing cell line resulted in similar editing efficiencies as the unmodified synthetic gRNAs, and cellular toxicity was observed with certain modification patterns. Of the modifications that were nontoxic, some patterns showed modest improvement in editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein. Overall, our results indicate that MS modifications are required for experiments with co-electroporation of Cas9 mRNA and synthetic gRNA, yet have no impact on editing efficiency when delivered with lipid-based transfection reagents.