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Chemical Modification of CRISPR <em>gRNAs</em> Eliminate type I Interferon Responses in Human Peripheral Blood Mononuclear Cells | OMICS International| Abstract
ISSN: 2576-3881

Journal of Cytokine Biology
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  • Research Article   
  • J Cytokine Biol, Vol 3(1): 121
  • DOI: 10.4172/2576-3881.1000121

Chemical Modification of CRISPR gRNAs Eliminate type I Interferon Responses in Human Peripheral Blood Mononuclear Cells

Mollie S. Schubert1, Edward Cedrone2, Barry Neun2, Mark A. Behlke1* and Marina A. Dobrovolskaia2*
1Integrated DNA Technologies, , Inc., 1710 Commercial Park, Coralville, IA 52241, USA
2Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, , Frederick, MD 21702, USA
*Corresponding Author (s) : Mark A. Behlke, Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA 52241, USA, Email: [email protected]
Marina A. Dobrovolskaia, Nanotechnology Characterization Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA, Email: [email protected]

Received Date: Jan 10, 2018 / Accepted Date: Jan 22, 2018 / Published Date: Jan 29, 2018

Abstract

Objectives: CRISPR/Cas9 is currently the primary tool used for genome editing in mammalian cells. To cleave and alter genomic DNA, both the Cas9 nuclease and a guide RNA (gRNA) must be present in the nucleus. One preferred method of introducing these reagents is direct transfection of a recombinant Cas9 protein complexed with a synthetic gRNA as a ribonucleoprotein (RNP) complex. It is well established from prior work in RNA interference that synthetic RNAs can induce a type I interferon (IFN) response that can limit the application of such methods both in vitro and in vivo. While the immunological properties of short siRNAs are well understood, little is known about the immune recognition of longer CRISPR gRNAs. The objective of our in vitro study was to investigate how the composition of the gRNA influences its recognition by human immune cells.
Methods: The study was performed in vitro in human peripheral blood mononuclear cells (PBMCs). The PBMCs from healthy donor volunteers were treated with gRNA for 24 h, and the levels of type I IFNs in culture supernatants were measured by a multiplex enzyme-linked immunosorbent chemiluminescent assay. Prior to the analysis in PBMCs, the physicochemical parameters and functionality of all nucleic acid constructs were confirmed by electrospray-ionization mass spectrometry and CRISPR/Cas9 gene editing assessment in HEK293-Cas9 cells, respectively.
Results: We found that unmodified synthetic CRISPR gRNAs triggered a strong IFN response in PBMC cultures in vitro that could be prevented with chemical modification. Likewise, in vitro–transcribed single-guide RNAs (sgRNAs) also triggered a strong IFN response that could only be partially suppressed by phosphatase removal of the 5’-triphosphate group. However, the process by which the gRNA is prepared (i.e., chemically synthesized as a two-part crRNA:tracrRNA complex or in vitro–transcribed as an sgRNA) does not directly influence the immune response to an unmodified gRNA. When experiments were performed in the HEK293 cells, only in vitro–transcribed sgRNA containing 5’-triphosphate induced IFN secretion.
Conclusion: The results of our structure–activity relationship study, therefore, suggest that chemical modifications commonly used to reduce the immunostimulation of traditional RNA therapeutics can also be used as effective tools to eliminate undesirable IFN responses to gRNAs.

Keywords: CRISPR/Cas9; sgRNA; gRNA; crRNA; tracrRNA; Interferons; Immunotoxicity

Citation: Schubert MS, Cedrone E, Neun B, Behlke MA, Dobrovolskaia MA, et al. (2018) Chemical Modification of CRISPR gRNAs Eliminate type I Interferon Responses in Human Peripheral Blood Mononuclear Cells. J Cytokine Biol 3: 121. Doi: 10.4172/2576-3881.1000121

Copyright: © 2018 Schubert MS, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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