Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity.

Megan Basila,Anja Van Brabant Smith,Melissa L Kelley,Stefan Maas

doi:10.1371/journal.pone.0188593

Abstract

Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.

Highlights

The class II CRISPR-Cas system is a bacterial adaptive defense mechanism that has been applied in mammalian cells for genome engineering [1,2,3,4]
We demonstrated that when the unmodified synthetic dual RNA system targeting PPIB was sequentially electroporated following Cas9 mRNA, gene editing was observable in K-562 cells, but in co-electroporation with Cas9 mRNA, gene editing was undetectable [8]
Upon sequential delivery of Cas9 mRNA and single guide RNA (sgRNA), an average of 51%, 28% and 25% indel formation was estimated for the same gene targets PPIB, PSMD7 and PSMD11, respectively, while co-electroporation resulted in a drastic reduction in indel formation to ~ 6%, 4% and 16%, respectively (S1 Fig)

Summary

Introduction

The class II CRISPR-Cas system is a bacterial adaptive defense mechanism that has been applied in mammalian cells for genome engineering [1,2,3,4]. The second RNA configuration uses a single guide RNA (sgRNA), which is comprised of both crRNA and tracrRNA fused together by a stem loop in the duplex region creating a long ~ 100-mer RNA [1] This chimeric strategy was employed to simplify the generation of expression plasmids or in vitro transcribed sgRNA molecules for editing in mammalian cells [6,7]. Both RNA configurations efficiently guide Cas to specific DNA sites in mammalian cells to cause a DSB [8]. Using the dual RNA system allows for rapid synthesis of gene-specific crRNAs that can be used with a universal tracrRNA, and, importantly, permits highthroughput generation of genome-scale libraries for arrayed screening applications [8,9,10,11,12]

Methods

Results

Conclusion