Applications of chemically modified synthetic guide RNA for CRISPR-Cas9 genome editing
Poster Jan 12, 2017
Megan Basila, Eldon Chou, Emily Anderson, Melissa L. Kelley, Anja van Brabant Smith
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.
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Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE
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The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s), phase II metabolizing enzymes and transporter genes in response to xenobiotics, including prescription drugs.READ MORE
Psychiatric Risk Gene Cacna1c and Early Life Stress: Potential Gene-Environment interactions?Poster
Early life stress (ELS) is highly associated with development of psychopathology
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